mancozeb (ISO); manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt  

Additional ecotoxological information

Administrative data

Endpoint:

additional ecotoxicological information

Type of information:

experimental study

Remarks:

aquatic outdoor mesocosm study (higher tier)

Study period:

1998-06-17 to 1999-10-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

other: assessment report

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: SETAC: Guidence Document on Testing Procedures for Pesticides in Freshwater Mesocosms

Version / remarks:

1991

Deviations:

yes

Principles of method if other than guideline:

The study was not performed in accordance with more recent guidance with respect to experimental design (treatments were not replicated. The analytical verification of the applied test article concentrations by measurements of Mancozeb was not performed. Further, minimum detectable differences (MDDs) for responses observed were not reported hampering the evaluation of the statistical power of the study.

The mesocosm study, however, had replicated controls and followed a nonreplicated regression experimental design with 7 treatment levels (nominal 1 – 1000 μg a.s./L). This mesocosm study was re-evaluated by Van Wijngaarden (2013) based on the procedure described in De Jong et al. (2008) (see section on "Overall remarks" below).

GLP compliance:

yes

Type of study / information:

Outdoor aquatic mesocosm study designed in accordance with the SETAC Guidence Document on Testing Procedures for Pesticides in Freshwater Mesocosms (SETAC, 1991) and performed following the principles of Good Laboratory Practice (GLP).

Test material

Test material information

Constituent 1

Reference substance name:

Complexation products of manganese and zinc with ethylenebis(dithiocarbamate)

EC Number:

616-995-5

Cas Number:

8018-01-7

Molecular formula:

(x+y)[C4H6N2S4]2- + xMn2+ + yZn2+, x:y ranges between 1:0.062 to 1:0.12 (mean 1:0.091)

IUPAC Name:

Complexation products of manganese and zinc with ethylenebis(dithiocarbamate)

Results and discussion

Any other information on results incl. tables

The folowing information refers to the study report of Memmert (1999) as summarized in the Renewal Assessment Report for Mancozeb, Volume 3 – B.9 (AS), prepared according to the Commission Regulation (EU) N° 1107/2009 and amended on March 2019.

 

Environmental conditions

Ambient air temperature, barometer readings, rainfall, relative air humidity, solar radiation and wind speed and direction were all measured and reported daily using a weather station located 200m from the mesocosms. As the recording was not performed in compliance with GLP, they have not been reported in the study report. However, the weather conditions (wind speed, air temperature, and whether or not it was raining) during application of the test substance were recorded and are presented in the table attached. Wind speed was low for every test substance application, though it rained on the 7th test substance application.

 

Analysis of ETU

Because of the rapid degradation of mancozeb in water, the main hydrolysis product ETU (Ethylenethiourea) was measured in the pond waters as a proxy for mancozeb concentration. Determination of ETU concentrations was carried out by HPLC/MS. This method only allows for the detection of freely dissolved ETU. To acquire the final measured concentrations of ETU, the two measurements taken on each sampling day were first averaged, then corrected for recovery after storage using a percentage derived from the experiment carried out on the storage stability of ETU (described in section I: E. of this summary). In the highest treatment concentration of Penncozeb 80 WP (1250 μg/L), levels of ETU were low during the first two weeks of the application period (maximum of 56 μg/L corrected average), increasing from day 23 to a maximum level of 2306 μg/L corrected average on day 51. The levels of ETU had decreased on day 105 (354 μg/L corrected average), 56 days after the final test substance application. For the lower test concentrations, measured levels of ETU were rarely above the limit of determination. A summary of the measured concentrations of ETU in experimental ponds is presented attached.

 

Degradation of mancozeb

The laboratory test on the degradation of mancozeb was carried out at pH 7.3 and 9.8. Assays with a nominal 1000 and 100μg/L were performed, but no reliable values for the measurement of mancozeb concentration were obtained from the 100μg/L assay due to the method of analysis. For the 1000μg/L assay at both pH used, the residual levels of mancozeb were not higher than 5% of the initial measured value after one week. For both the 100 and 1000μg mancozeb/L assays performed at pH 9.8, and the 100μg/L assay performed at pH 7.3, the measured levels of ETU increased between the measurements made at 24 hours and 7 days. However, the measurements of ETU concentration made in the 1000μg/L assay performed at pH 7.3 were lower after 7 days than they were after 24 hours. Thus, the lab study performed on the degradation of mancozeb has provided no clear relationship between mancozeb degradation and ETU concentrations in the pond water system. Because of this, and as no measurements of mancozeb could be made at the lower concentration, no conclusions can be made on the presence of mancozeb based on the measurements of ETU made during the mesocosm study. The results from this study are presented in the table attached.

 

Biological effects- Zooplankton

No measurements of mancozeb concentration in the mesocosms were made throughout the test, nor was a relationship between mancozeb concentration and ETU concentration determined in the lab studies (see section II.C). Despite this, all endpoints are based on nominal concentrations of mancozeb.

 

Community Measures

Statistical analysis revealed that Penncozeb 80 WP statistically significantly reduced the taxa abundance of zooplankton during the treatment period, an effect that increased with increasing test substance concentration. The calculated EC20 and EC50 were 55 and 3109 μg product/L respectively. In addition, a significant dose-response relationship in diversity was found using the Shannon-Weaver analysis. There was a reduction in diversity by the test article. The calculated EC20 was 256 μg product/L, however an EC50 could not be calculated as effects did not reach 50%. No significant trends in taxa abundance were found for the pre-treatment or post-treatment periods, and there was no statistically significant dose-response relationship on evenness detected during the treatment period. Statistical analysis of similarity demonstrated significant dose-response relationships for both the Steinhaus and Stander similarity indices. Between similarity of mean control and each treatment was highest at the first application and reduced over time- potentially indicating that Penncozeb 80 WP treatment reduces the similarity between treatments and control. However, the same trend occurred at the within similarity of the 3 control replicates, therefore this trend may simply be the product of changing population dynamics over time, regardless of test treatment.

 

Population Dynamics

Only the most dominant zooplankton species were studied for detailed population dynamics.

 

Crustaceans

The crustacean zooplankton comprised mainly of cladocerans and copepods. Marked reductions of crustacean numbers were observed at the highest test concentration, until the post-treatment period. There was also a decline in abundance in the abundance of the species Chydorus sphericus and Nauplii sp. during the late treatment period and post-treatment periods. This trend was followed also by the control. Decline in control densities to treatment levels is not considered true recovery.

 

Rotatoria

The species of Rotatoria found in sufficient enough densities for the evaluation of toxic effects were Branchionus leydigi, Keratella quadrata, Hexarthra spec. and Cephalodella spec. The first three of these species (which were the most dominant) as well as the sum of all rotatoria showed statistically significant density reductions from the test concentration of 40μg/L, and there were statistically significant dose-response relationships.

 

Biological effects- Macrozoobenthos

Community Measures

The majority of insects and larvae collected by MASS were chironomids. Low values of abundance and diversity made trends difficult to identify, and the only statistically significant trend for the treatment period was for taxa abundance. In addition, significant concentration-dependant reductions in diversity and evenness were found during the post-treatment period.  The statistical analysis of similarity indices showed statistically-significant dose-response relationships during the treatment and post-treatment periods. The highest test concentration demonstrated an observable reduction in between similarity (with the mean of the control replicates).

 

Population Dynamics- Emergence Traps

Chironomus spec.

By day 14, the numbers of emerging Chironomus almost reached 0 at the highest test substance concentration. This rapid drop was not followed by any recovery, which could be the result of the population producing the overwintering developmental phases, as well as the particularly low levels of periphyton found in the highest test concentration.

 

Culicidae

Levels of Culex spec. larvae increased at the two highest test concentrations during the post- treatment period, showing rapid and over-compensating recovery. This may be due to a drop in competition to the larvae from zooplankton during the treatment period, as well as higher levels of phytoplankton. It is assumed that, as rates only increased following the treatment period, the test substance concentrations were subduing numbers of Culex during the treatment period.

The only statistically significant change in population dynamics was seen in Culex species.

 

Population Dynamics- MASS

Few species were found in any great numbers using the MASS sampling; only Chironomus spec., oligochaetes, and Chaoborus crystallinus (only in the post-treatment period). The inhibition of Chaoborus crystallinus development during the treatment period may have been caused by direct toxicity of the test substance, or by indirect effects such as a lack of rotifers as a food source. No statistically significant effects of test substance on MASS-sampled organisms were found, and no ECx values were calculated due to the low sampling rate. There were no observable dose-response relationships, save for potentially the Chaoborus crystallinus larvae; however it is not possible to determine if low numbers of Chaoborus crystallinus was a dose effect due to the lack of sampling before the treatment period.

 

Biological effects- Phytoplankton

Community measures

A large number of phytoplankton taxa were identified in the mesocosms. There was no statistically significant relationship between taxa abundance and test substance in the pre- and during treatment periods. However, there was a significant relationship found in the post-treatment period. There was a statistically significant dose-response relationship on diversity and evenness at the two highest treatment concentrations during the treatment periods. The EC20 and EC50 for diversity were 142 and 530 μg product/L. The EC20 and EC50 for evenness following treatment were 364 and 417 μg product/L.  Statistically significant dose-response relationships were identified for the Stander’s and Steinhaus’ indices of similarity. Changes in the abundance of Gloeocystis and Ankyra were the biggest contributors to differences in similarity between the control and dosed treatments

 

 

Phytoplankton Biomass (Chlorophyll-a)

The concentrations of chlorophyll-a was used as a total phytoplankton biomass estimate. Phytoplankton biomass increased at the highest test concentration during the treatment period. The second highest peak during the treatment period was observed in the second highest test concentration.

 

Population Dynamics

Some phytoplankton species demonstrated no sensitivity to the test substance, and in fact increased in cell density with an increase in test substance concentration. This is expected to be an indirect effect resulting from lack of grazing by zooplankton. Species which demonstrated this positive dose-response relationship included the dominant species Gloeocystis, Hyaloraphidium and Scenedesmus as well as large Cryptophytes and Chlamydomonas spec.  The phytoplankton species Kirchneriella, Ankyra, small Cryptophytes and coccale Chlorophyceae all demonstrated no statistically significant dose-response relationships. They appeared to have no positive or negative, direct or indirect responses to test concentration.  One species, Volvox, showed a negative dose-response relationship, with cell density reducing with increasing test substance concentration. EC20 and EC50s were calculated for the inhibition of Volvox.

 

Biological effects- Periphyton

Periphyton biomass was determined using the measured parameters of Chlorophyll-a and Dry weight. These parameters were measured on test days 0, 56, and 77. There were no statistically significant dose-response relationships with either chlorophyll-a or dry weight.

Applicant's summary and conclusion

Conclusions:

In an outdoor semi-field mesocosm study by Memmert (1999) as re-assessed by van Wijngaard (2013) a NOECpopulation of 3.2 μg a.s./L and a NOECcommunity of 10 μg a.s./L was derived. The study is regarded reliable and sufficient for assessment.

Executive summary:

To investigate the potential impact of Penncozeb RT 80WP (a.s. Mancozeb) on aquatic ecosystems Memmert (1999) carried out an outdoor semi-field mesocosm study. The applied study design followed the Guidance Document on Testing Procedures for Pesticides in Freshwater Mesocosms. Proceedings from the workshop “A meeting of experts on guidelines for static field mesocosm tests”, Monks Wood, Huntingdon, UK, 3-4 July 1991. This study focussed on phyto- and zooplankton communities and benthic organisms. The reliability and results of this study were re-assessed by van Wijngaard (2013). The observed treatment-related responses in the mesocosms were re-evaluated by using "Effect classes" after De Jong et al. (2008) and the EU Guidance Document on Aquatic Ecotoxicology (SANCO 2002). The test location was at Aachen University of Technology Dept. of Biology V (Ecology, Ecotoxicology, Ecochemistry), Aachen, Germany. The study took place from June to September 1998. The test systems consisted of 10 artificial ponds (glass-fibre reinforced polyester tank; diameter approx. 2 m; 2 m depth; 5000 L water volume (1.6 m water layer, 10 cm sediment layer). Sediment and biota were obtained from local non-polluted natural ponds. Macrophytes were not introduced. The ponds were filled with local tap water. Sediment and biota were introduced in March 1998. The following test concentrations were used: 1.25, 12.5, 40, 125, 400 and 1250 μg/L Penncozeb 80 WP. This corresponds to nominal: 1.0 μg Mancozeb/L; 3.2 μg Mancozeb/L; 10 μg Mancozeb/L; 32 μg Mancozeb/L; 100 μg Mancozeb/L; 320 μg Mancozeb/L; 1000 μg Mancozeb/L. Number of replicates: controls n=3; treatments n= 1 (regression design). A spray application method was chosen to simulate the entry of the test article into a water body by direct overspray or spray drift. Eight separate, identical applications of the test article were made to each pond. The controls were treated with deionized water only. Time interval was 7 d between applications (June 17 and 24, July 01, 08, 15, 22 and 29, and August 05, 1998). Applications were performed in the early afternoon (between 13.00 and 15.00 h). The test item was applied by means of a hand-held spray boom. Dynamics of the test compound in the water were monitored as the main hydrolysis product ETU (ethylenethiourea).

Water samples for biological characterisation were collected by means of a depth-integrated sampling technique. At each sampling date six independent water samples were taken from defined locations in each pond. Sampling points were identical at all sampling dates. All six samples per pond were combined (altogether about 60 L per pond). Then subsamples were withdrawn from this 60-L sample for biological determinations and chemical analyses of the water. The remaining water was returned to the corresponding pond.

Phytoplankton: For the taxonomic identification and counting of phytoplankton organisms a subsample of about 250 mL was preserved. Population densities were presented as individuals per water volume. Chlorophyll a: For the determination of Chl-a concentrations, 0.15 - 4 L water was filtered through a Whatman GF/C filter. Filters were extracted with ethanol and the extracts were processed to determine phytoplankton Chl-a photo-metrically.

Periphyton: glass slides (26x76.5 mm) were used as artificial substrate. Three of the six slides from each sampling date were used for triplicate dry weight measurements. For Chl-a measurements the periphyton was quantitatively wiped off with a Whatman GF/C microfiber glass filter and extracted. In case of a low periphyton density the filters with periphyton of two or all three glass slides were combined to one sample. The extraction and measurement of Chl-a was similarly performed as for phytoplankton.

Zooplankton: A defined water sample (2 – 15 L) of the depth-integrated sample was filtered through a 55 μm gauze. Counting and identification was performed by means of an inverted microscope. Population densities were presented as individuals per water volume.

 

Emerging insects: Insects were collected weekly with floating emergence traps (0.25 m2 base).

Macroinvertebrates: The occurrence of macroinvertebrates was recorded by using artificial substrates. Population densities were reported as individuals per duplicate artificial substrate.

Covering the complete study period, the mean water temperature over all microcosms ranged from ca. 15 – 23 °C at a depth of 20 cm. At a depth of 140 cm the temperature ranged from ca. 15 – 20 °C. Over the study period, the pH was around 7.1 – 8.9 in the controls, with little difference between the depth of 20 and 140 cm.

This also accounted for the electric conductivity which ranged from some 270 to 300 μS/cm during the complete study In the controls, at 20 cm, dissolved oxygen (DO) concentrations normally were between 2 and 6 mg/L. Lowest values were around 1 mg/L occurring on a few occasions and replicates by the end of the experiment. At 140 cm, DO concentrations also generally were between 2 and 6 mg/L. The lowest concentrations of < 1 mg/L occurred on Day 7 in all three controls (and other test systems). The active substance Mancozeb is not stable in water systems and quickly degrades within hours (e.g. oxidative processes and hydrolysis). Reported hydrolysis half-life periods are from 2.2 h for acidic pH to 14.1 h for alkaline pH. Therefore, only the actual concentrations of the main hydrolysis product ETU were monitored in the water. ETU residues in water (depth-integrated) were followed over time (Table A). During the sampling procedure different locations in the test systems were sampled. Analytical results were obtained by a HPLC/MS method. The limit of detection for the samples of the microcosm study varied between about 1.7 and 3.4 μg ETU/L. Water temperature, pH, dissolved oxygen (DO), and electrical conductivity (EC) were measured according to the study schedule. Nutrients, total organic carbon and water hardness were also measured at regular intervals.

Based on the re-assessment by van Wijngaarden (2013), it is concluded that the rotifers were the most sensitive group. A NOECpopulation of 3.2 μg a.s./L is consdered for the most sensitive taxa (the rotifer Hexarthra sp. and the green alga Volvox). The lowest NOECcommunity of 10 μg a.s./L is derived for the zooplankton community. The study is regarded reliable and sufficient for assessment.