Phytotoxicological assessment of AMD affected river waters in “Sredna Gora”, Bulgaria to Lepidium sativum L.

The ongoing pollution of water bodies from decommissioned mines and mining waste facilities in Bulgaria is a serious problem, leading to a contamination of river waters with heavy metals. Although the chemical analyses of acid mine drainage (AMD) affected waters provide information about their toxicity, this information is not enough to evaluate the potential impacts on the biota. For this, different biological responses are used to assess the toxicity of the polluted waters. The goal of the research was to determine the phytotoxicity (using Lepidium sativum) of five affected river waters. The lower reaches of the rivers flow though agricultural areas and are often used for irrigation by local farmers, increasing the chance of produce contamination. The effect of the working solutions on seed germination and primary root growth of L. sativum was assessed by the following indices: Relative seed germination (RSG), relative root growth (RRG) and germination index (GI, the product of RSG and RRG). The obtained results have shown the connection between germination/root growth inhibition and concentrations of heavy metals in working solutions, but there is no connection between germination/root growth inhibition and pH levels.


Introduction:
Physicochemical parameters can be used to characterize the water, but do not provide enough information to ensure that surface or ground water is environmentally safe for agricultural use [1;2].
Knowledge of the toxic effects of pollutants is a key requirement for environmental risk assessments.A fundamental instrument in this field is given by ecotoxicological tests, which use organisms that belong to different trophic levels and involve distinct endpoints, targets and modes of action.These bioassays can be used either individually or as components of a test battery, which is highly recommended when evaluating the toxic effects of unknown substances, mixtures of chemicals and samples of complex composition, e.g., AMD contaminated waters [3].
In recent years, there has been a significant increase in the studies of the effects of mining pollution to the aquatic organisms, but most of the studies represent toxicity effect over the aquatic faunafishes and aquatic invertebrates [4;5].Other studies focus on plants, as members of the food chains, for they pose risk to humans and animals alike through contamination of food supplies [6;7].
Plants show a wide variation in sensitivity to metal contamination.Some of them are hyperaccumulators while others display great sensitivity to pollutants [8].There are many publications which aim is to determine the best plant test-objects with regard to their sensitivity to different pollutants, such as heavy metals.Many authors point Lepidum sativum L. as one of the most sensitive species, among commonly used in phytotoxicological tests, to the metal (oids) of Cu, Zn, Pb and Hg [9].
The phytotoxicological biotests have many advantages: they are cheap and easy to use, do not require expensive laboratory equipment, are easy to observe, and produce repeatable results [10].
Currently, in Bulgaria there are many publications, related to the impact of mining industry on the environment and human health.Most of the studies focus on hydrogeological and physicochemical parameters of the studied regions, assessment of background concentrations, lithochemical anomalies and pollution in the watersheds [11;12;13;14;15;16]; influence of point and diffuse sources of 1305 (2024) 012010 IOP Publishing doi:10.1088/1755-1315/1305/1/012010 2 pollution on water intakes [17;18;19;20]; bioaccumulation of heavy metals in biota [21;22;23]; effects of heavy metal contamination on specific test organismsfishes and invertebrates [24;25].
So far, we have not found any previous studies on phytotoxicological assessment of AMD affected river waters to Lepidium sativum L. in Bulgaria.According to the official information from the Ministry of Energy there are 488 closed, mine waste facilities, including abandoned mining sites in Bulgaria [26], which are potential sources of pollution to the local watercourses.The affected rivers are often used for irrigation, increasing the chance of produce contamination.In order to assess the influence of the closed facilities, we have selected several affected rivers in the mining district of "Sredna Gora" mountains.Some of the rivers are in watersheds with active mining, while others are draining their waters from closed and abandoned mining facilities, like mines, embankments and tailing ponds.
The aim of our research was to compare the impact of working vs. closed mining facilities on the adjacent rivers by toxicity tests with the garden cress L. sativum L. In order to assess the impact of AMD on the rivers we have collected samples at both high and low rivers discharge.The data supplements the information provided by previous studies on the pollution in the region, and provides additional informational for the effects of recultivation of mining cites and their environmental impact in the context of climate change.

Study sites
Twelve sampling stations were selected: six around former mine "Elshitsa" in the watershed of Elshishka River, two around former open-pit mine "Medet"on Medetska River and Topolnitsa River, and four others in streams around working open-pit mine complex "Asarel Medet"in the Banska Luda Yana River watershed.Table 1 presents some general characteristics of the sampling stations, and Figure 1 gives the relative positions of the sampling stations.As the mining facilities are located in mostly in separate watersheds, cumulative effect of pollution is observed after the confluence of Medetska into Topolnitsa River.Only one station can be taken as a reference station -EUV, located above the village of Elshitsa.The EBV appeared to have no AMD or significant household influences, although it suffered from insufficient flow in summer.Six of the seven sites around Elshitsa mine were AMD or influenced at different degree by the AMD river waters.In the region of Asarel Medet the produce waters, drainage waters from the waste dumps (embankments) and the tailing pond are treated in waste water treatment plants before subsequent release into the rivers.Thus, Mechinska and Asarelska rivers are influenced by WWTP effluents, while Panova River is affected by underground acid drainage from the situated in close proximity, both, "Lyulyakovitsa" tailing pond, and open-pit mine.Medetska River, below Medet mine, is chronically impacted by AMD from the open-pit mine lake and several waste dumps, and Topolnitsa Riverby the largest tailing pond on the Balkan peninsula, containing 170 000 000 m 3 of waste material.
The samples were collected in the period from May to November, 2023 with different monitoring frequency for Elshitsa mine complex, which was sampled one week after the Medet mine region.Data on the amount of heavy metals in the AMD and river waters was kindly provided by the corresponding river basin directorate (river water quality data) and by the company responsible for the planning and execution of the technical liquidation, recultivation, water purification and monitoring of waters in the vicinity of closed mines.

Phytotoxicity assessment
The phytotoxicity tests of the AMD and the river waters were performed using the garden cress Lepidium sativum L. to assess the inhibition of seed germination and root and shoot growth.The results were determined after 7 days of exposure to the test waters in the dark in room temperature.The germination tests were performed in petri dishes (15 cm diameter) lined with filter paper, with each dish containing 20 seeds of cress.The seeds were pre-drenched in tap water.For each sample, as for the control, 3 replicates were performed (giving the total of 60 seeds used).After the incubation period, germinated seeds were counted and the root length was manually measured to the nearest 1 mm.A visible root was used as the operational definition of seed germination.
All experiments with AMD samples and with the most heavily affected river waters were conducted, both, with unaltered water samples and with samples diluted 1:10 with distilled water.
Three parameters were measured in the phytotoxicity bioassay: relative seed germination (RSG, eq.1), relative root growth (RRG, eq.2).Based on those parameters germination index (GI, eq. 3) was calculated according to [27].The advantage of the GI is that it allows the evaluation of low toxicity (affects root growth) and high toxicity (affects germination): RSG % = Gs/Gc x 100 (1) RRG % = LRs/LRc x 100 (2) GI % = RSG x RRG/ 100 (3) where: Gs and Gc are the number of germinated seeds in the sample and the control treatment, respectively; LRs and LRc are the root lengths of the germinated seeds in the sample and in the control, respectively.
In general, GI values within the range of 90% and 110% represent "no significant effect", values of GI less than 90% indicate inhibition, and higher than 110%stimulation [28,29].Aguerre and Gavazzo and Manas and De las Heras [30] further divide the scale with the following values: GI above 80% slight inhibition; below 50%strong inhibition and between 50 and 80% indicate moderate inhibition.
Another system is proposed by Persoone et al. [31]  This system can by directly applied to all of the above-mentioned parameters as they are expressed as percentage effects (PEs) of germination and growth inhibition.It is useful for the categorization and comparison of different samples based on the observed effects, regardless of the type of pollutants behind the toxicity effects [32].After determining the percent effect for each biotest, the sample was classified as one of five classes according to the highest toxicity indicated by at least one test.

Water quality parameters
The lowest average pH values of 2.12 were measured in the AMD filled Red Lakestation RL (Table 3).All stations below RL show pH values below 3 (stations KDR and EUA).The station below the south waste dump (embankment) of Elshitsa mine is also characterized with low pH values (3.21), but the AMD generated from the waste dump heap is not as acidic as the one seeping out of the mine.Apart from the pH values, the AMD from the dump heap differed in color, as it was clear, compared to the reddish color of the AMD from the mine.The pH of the reference station for Elshishka Riverupstream of the village (EUV) is 7.89, remained relatively stable after passing through the village (7.76) and dropped sharply after the confluence of AMD impacted Kamen dol Creek (KDR).The pH values in the upper part of the Banska Luda Yana watershed consistently had the highest pH are all above the neutral point.Mechinska River at RMC consistently had the highest pH -8.05, while the lowest was measured in Panova River (7.0).Although the pH is higher, it drops to lowest of 4.54 in Panova River after a late spring rains that may have increased the amount of generated AMD.Medetska River at RMD had average pH values below 4, whish slightly increased after its confluence into Topolnitsa River (5.29).
Except for the RL the DO values were always at or near saturation, and will not be discussed further.The pH generally decreased throughout the summer, during the low flow of the rivers.Conductivity followed a similar trend to pH, i.e. stations with low pH measurements had high conductivity (Table 3).For example, the average conductivity was highest at SE (10775 μS/cm) and ranged from 10400 to 11150 μS/cm.The conductivity in the RL station is lower than the measured at SE, probably due to precipitation of hydroxides in the lake itself.The precipitation continues below the lake and slightly lowers the conductivity at Kamen dol Creek at KDR.The conductivity of Elshishka River at EUV station is 366 and increases to 2609 after the inflow of KDR and the rest of small AMD creeks and seepage from the tailing pond.Conductivity was lowest at PR (276 to 375 μS/cm).The general relationship between pH and conductivity is not valid in MCR and AR, both receiving inflows from the WWTP of the active metallurgical complex.The purification of the process waters increases, both, the pH and conductivity of the effluents, and subsequently of the river waters.For those rivers [24] report moderate ecological potential based on the macrozoobentic communities.As there are no other anthropogenic influences, the slight decrease in the water quality is related to the occasional increases of heavy metals in the river waters, due to incidental releases/overflows of industrial process waters through the WWTPs.The conductivity values in Medetska River at RMD are typical for AMD impacted rivers.The higher discharge in Topolnitsa River dilutes the inflowing waters to an average value of 901 μS/cm.The values of the measured parameters in the phytotoxicity bioassay are given in Table 4, and in Table 5, are given the relative orders of the sampling stations, according to the values of the parameters.We have expected that the increased conductivity (amount of dissolved substances) of water and the expected higher concentrations of heavy metals would decreased seed germination, as noted in previous studies [33;34].Contrary to that, the seed germination was higher in the samples affected by AMD from the closed mines.At those sampling stations the RSG was hardly inhibited (RT, RMD and RL) or even showed better germination than the control seeds (KDR and EUA), but with no significant effect.The only station from this group which shows signs of inhibition was the AMD below the south embankment of the closed Elshitsa mine (SE).Only in the case of RL we observed black coloring of the seeds, which according to [35] is a sign of accumulation of metals from the solution.The samples from the region of the working metallurgical complex showed, in general, up to 5% reduction in the RSG with no significant effect.Panova River at RP had RSG of 83.87%, which is indicative of inhibition and is one of the lowest values for this parameter in our study.The river is influenced by diffuse AMD, through underground seepage, presumably, from the Lyulyakovitsa tailing pond.Occasionally, RP shows increased precipitation of hydroxides with rusty coloration of the bottom sediments.The river selfpurification by sedimentation of the hydroxides determines the negligible effect on RA.The other station with a peculiar result is the control station on Elshishka River, situated upstream the village of Elshitsa (EUV), as it showed the strongest inhibition of the seed germination with RSG values of 82%.Although the station is not expected to be affected by AMD, in its watershed are situated the arable lands of the village, so the inhibited germination of the seeds may be due to agricultural pollution, rather than AMD.

Relative root growth and germination index.
The values of RRG show different and more logical response to experimental design (Table 4).The order in mean RRG for the studied samples was reversed, with the stations in the region of operational mining facilities and those with no expected AMD influence moved to the left and those in the region of closed mining facilitiesto the right (Table 5).
RRG exceeded 100% only at RP station, thus Panova River shows both the lowest RSG and the highest RRG in the study.The RRG shows very good relation to the AMD influenced stations, with lowest values in RL, RMD and SE.Thus, the two groups are clearly distinguished with all samples in the vicinity of operational mines showing less than 20% reduction, and those in the regions of abandoned minesgreater than approximately 35% reduction in RRG.The results for GI resembled those of RRG to a large extent, except for some stations that exchanged their order, but stayed in the same group (Table 5).All samples from the working mines group have GI values higher than approximately 80%, suggesting slight or no inhibition of the growth of L. sativum.The group around the abandoned mines had GI values lower than 50% (strong inhibition), with only Elshishka River at EUA, showing moderate inhibition (GI ≈70%), indication selfpurification along the river.

Toxicity classification
According to the toxicity classification system of Persoone et al. (2003) [31] the examined waters range from no significant toxicity (class I) to significant toxicity (class III) of the sample waters Table 6.The results for the toxicity of the sample waters divide them at three groups according to the acute hazard they pose.All of the samples in the no hazard group are from the region of operational mines, while in the acute hazard group there are only abandoned mines river-and AMD cites.The II toxicity class (low hazard group) consists of sampling stations with reduced effects from the mining industry, due to the influence of WWTP, river selfpurification, or to supposed influence by pollutants from agriculture.

Conclusions
It is apparent that cress seeds (Lepidium sativum L.) are quite sensitive in phytotoxicological analysis of AMD affected river waters.The seeds reaction is precise enough so the tests can be used in the regular monitoring of the mine impacted river waters.Our study shoes the importance of post closure operations for the prevention and control of AMD pollution.
Climatic conditions, in particular the amount of rain and river discharge, significantly influence the results of the tests, as they alter the AMD generation and concentration in rivers.During high river flow the GI values approach 100 %, and with decreasing discharge GI fall below 30 %, while prolonged rains caused the increased dissolution and leaching of contaminants from the embankment increasing the strength of the generated AMD.
Our research continues, and in the upcoming season, our focus will be on validating the established results.This ongoing effort is crucial for reinforcing the reliability of our findings and ensuring the robustness of our conclusions.

References
Seed emergence and growth parameters 5.2.1.Relative seed germination.The aim of our research was to compare the impact of working vs. closed mining facilities on the adjacent rivers by toxicity tests with the garden cress L. sativum L.

Table 1 .
General description of the sampling stations.

Table 2 .
Average values for the summer period of some metals in the River water at the corresponding sampling stations.Permission for the field study, conducted on public land was not provided, because water samples collected during the research were tested in laboratory.The species used in bioassays are commonly used laboratorian test objects.Field measurements of selected water quality parameters were made on each sampling date (around Elshitsa mine: 02.06; 06.07; 12.08.2023; around Medet and Asarel Medet: 29.06; 31.07;12.08; 03.10; 03.11.2023) at 12 sampling stations.The pH and conductivity measurements were made using HI 98129 pH&EC combo meter.Dissolved oxygen (DO) was measured by WTW Oxi 3310.All instruments were calibrated prior to the field measurements.
a Maximum allowable concentrations (MAC) and annual average values (AAV).AAV are given in italics (Ordinance No. H-4).MAC values for Al are pH dependent -<6.5 pН>6.5

Table 3 .
Average values of the measured water quality parameters at the corresponding stations.

Table 4 .
Mean values of the measured parameters in the phytotoxicity bioassay.

Table 5 .
Ordination of the sampling stations according to the values in Table4.Values decrease from left to right.In gray are given the stations in the region of closed mining facilities, in orangein the region of operational mining facilities, and the samples with no expected AMD influence are left blank.

Table 6 .
Toxicity of sample waters.