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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Key result

Duration:

77 d

Dose descriptor:

NOEC

Effect conc.:

10 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: population dynamic

Remarks on result:

other: based on effects of eight weekly short-lived pulses of Penncozeb 80 WP on zoo- and phytoplankton communities and macrozoobenthos on the community level

Key result

Duration:

77 d

Dose descriptor:

other: NOEAEC

Effect conc.:

< 10 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: population dynamic

Remarks on result:

other: based on effects of eight weekly short-lived pulses of Penncozeb 80 WP on zoo- and phytoplankton communities and macrozoobenthos

Key result

Duration:

77 d

Dose descriptor:

NOEC

Effect conc.:

100 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: population dynamic

Remarks on result:

other: NOEC derived for phytoplankton community level

Details on results:

The NOEAEC for this study is set at < 10 μg a.s./L because:

- at the 10 μg a.s./L-treatment level, pronounced long-term reductions without recovery (Class 5B-effect) occurred in the most sensitive endpoint (i.e., the rotifer Hexarthra sp.).

- between the concentration of 3.2 μg a.s./L (Class 1-effects only) and 10 μg a.s./L (Class 5B-effects) no intermediate concentrations showing Class 3A-effects were available.

- at concentrations below 10 μg a.s./L (1.0 and 3.2 μg a.s./L), no effects were
observed.

- at the 10 μg a.s./L-treatment level, the impact on the most sensitive phytoplankton species (Volvox sp.) was short-term only.

- at 10 μg a.s./L direct effects within the most sensitive group (rotifers) were not observed, except for Hexarthra sp. which is one of the 11 rotifers monitored in the study.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th April to 10th April 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

other: ASTM. 1984 "Proposed Standard Practice for Conducting 96 Hour Tox- icity Tests with Microalgae. ASTM Committee E-47.01, Draft No. 7. 25 p.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 122-2 (Algal Toxicity, Tiers I and II)

Version / remarks:

U.S. Environmental Protection Agency. 1983. Environmental Effects Test Guidelines, Freshwater and Marine Algae Acute Toxicity Test. EPA 560/6-82-002, EG-22, October, 1983.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA 40 CFR, Part 158

Version / remarks:

U.S. Environmental Protection Agency. 1984. Data Requirements for Pesticide Registration; Final Rule. (40 CFR, Part 158). Federal Register Vol. 49 (No. 207), October 24, 1984, 158.150.

Deviations:

no

Principles of method if other than guideline:

The study was conducted according to US. EPA 1984 and ASTM 1984 standard procedures for the acute toxicity testing of freshwater green algae, which is equivalent to EEC method C3, except that duration of the study was 120 hours instead of 72 hours.
In accordance with Regulation (EU) No 1107/2009 the study was peer reviewed by rapporteur member states and have been proven to be valid by the EFSA.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analysis of algae culture medium samples for mancozeb at 0, 48 and 120-hours of testing was accomplished following the procedure described for the analytical method.
The samples from the algae toxicity test were prepared in the following manner:
At the 0-hour sampling, 15 mL aliquots of test media were collected directly from volumetric flasks containing each test concentration immediately following preparation.
At the 48-hour sampling, 15 mL aliquots of test media were collected from replicate "D" (an extra replicate) of each test concentration.
The 15 mL sample at 120-hours was composited from replicates A, B and C of each test concentration. Fortification of quality control samples with concentrations bracketing the nominal test concentrations were made after collection and preparation of the test samples. The samples were analyzed from mancozeb using the operating parameters described for the GC analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test concentrations were prepared directly in 1000 mL volumetric flasks as proportional dilutions of a 2000 mL solution of the highest test concentration of 0.50 mg/L. The stock solution was prepared by pipetting an appropriate aliquot of a primary standard into the sterile medium. Each treatment level and the control were prepared in quadruplicate using 100 mL of the appropriate concentration for each test vessel. The fourth replicate was used for the analytical verification at 48 h.
- The test media prepared just before the start of the test.
- Controls: Three replicates (without test item) were exposed under the same conditions as the test concentrations.
- Replicates per test concentration: 3
- Replicates of the control: 3
- Evidence of undissolved material: no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: Printz
- Source: Department of Botany, Culture Collection of Algae, Uniersity of Texas at Austin, Texas 78713-7640 (supplier batch: #1648, ABC culture #J4)
- Cultivation: at ABC Laboratories under standardized conditions
- Inoculum culture: set up two days before the start of the exposure
- Nominal algal cell density in test solutions: 3E03 cells/mL (determined using hemacytometer and an Olympus microscope)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

120 h

Test temperature:

24 ± 1 °C

pH:

at 0 h: 7.7 to 7.8
at 120 h: 7.3 to 8.1

Dissolved oxygen:

not reported

Nominal and measured concentrations:

Nominal: 33, 65, 130, 250 and 500 µg a.s./L,
Measured: 22.2, 42, 96, 174 and 376 µg a.s./L

Details on test conditions:

- Test vessel: Erlenmeyer flasks, volume: 250 mL
- Fill volume: 100 mL
- Initial cells density: 3E03 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Agitation: Test containers were placed on an orbital shaker

GROWTH MEDIUM
- Standard medium used: Yes, AAP Medium (see table in section "Any other information on materials and methods incl. tables")

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was composed of 1.0 mL of each of the nutrient solutions (A and B) diluted to a final volume of 1000 mL of autoclaved reverse-osmosis water. After preparation, medium was adjusted to pH 7.5 ± 0.1 and re-sterilised by passage through 0.45 μm filter.

EFFECT PARAMETERS MEASURED Cell counts were conducted at 24, 48, 72, 96, and 120 hours. Initial counts were performed on control replicates.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Yes (to 7.5 ± 0.1)
- Photoperiod: 24 h/day
- Light intensity and quality: Cool-white fluorescent light (400 ± 10 % ft-c, approx. 4300 lux)
- Temperature: Test solution were kept at 24 ± 1 °C.

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

8.79 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

120 h

Dose descriptor:

EC20

Effect conc.:

4.48 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Duration:

120 h

Dose descriptor:

EC10

Effect conc.:

3.54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

yield

Key result

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

32.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

120 h

Dose descriptor:

EC20

Effect conc.:

14 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

120 h

Dose descriptor:

EC10

Effect conc.:

9.05 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Any stimulation of growth found in any treatment: No
- Any observations that might cause a difference between measured and nominal values: No
- Measured concentrations: The measured concentrations of the test item during the exposure interval were in the range between 67 and 80 % of the nominal values at the start of the test, 17 to 20 % at 48 h of exposure and 1.3 to 7.3% at the end of the test.
- Effect concentrations exceeding solubility of substance in test medium: yes

Reported statistics and error estimates:

Cell counts for each concentration and control were subjected to analysis of variance (ANOVA) and treatments means were compared using a multiple means test (Dunnett's).

In the study report results are reported based on nominal concentrations. Since the levels of measured Mancozeb were well below 80% of the nominal by the end of the study, nominal endpoints are not acceptable for use in risk assessment and the validity of this study was questioned by Rapporteur Member State (RMS). The applicant provided a re-evaluation of the analytical data and EC50 and NOEC values based on the geometric mean of the active substance concentrations measured at 0, 48 and 120 h. These calculations were considered acceptable by the RMS and EFSA (EFSA Conclusion on the active substance Mancozep - Peer review of the pesticide risk assessment of the active substance mancozeb; European Food Safety Authority (EFSA), approved 12 June 2019, doi:10.2903/ j.efsa.2019.5755).

 

Table 1: Analytical data and results of their re-evaluation (RAR Mancozeb Vol. 3CA B9, 2018)

Time [h]	Nominal concentration [µg a.s./]
	33	65	130	250	500
	Measured concentration [µg a.s./L]
0	22.2	42	96	174	376
48	5.66	11.8	25.3	51.5	99.6
120	0.432	1.01	4.68	18.3	36.3
Geomean	3.79	7.94	22.5	54.7	110.8

 

The following ECx re-calculations were considered acceptable by the RMS and EFSA (EFSA Conclusion on the active substance Mancozep (Peer review of the pesticide risk assessment of the active substance mancozeb; European Food Safety Authority (EFSA), approved 12 June 2019, doi:10.2903/ j.efsa.2019.5755)).

 

Table 2: ECx values based on geometric mean measured concentrations (re-evaluation, RAR Mancozeb Vol. 3CA B9, 2018)

Endpoint	Effect concentration [µg a.s./L] (95% confidence intervals)
	NOEC	LOEC	EC10	EC20	EC50
Growth rate	n.d.	n.d.	9.05 (8.98 – 9.11)	14.0 (13.9 – 14.1)	32.2 (32.1 – 32.4)
Yield	n.d.	n.d.	3.54 (3.02 - 4.15)	4.84 (4.38 – 5.34)	8.79 (7.79 – 9.91)
n.d.: not determined

The EC50 of 32.1 µg a.s./L based on the growth rate (mean measured) is considered acceptable for the use in the risk assessment.

Validity criteria fulfilled:

yes

Conclusions:

The test item had an inhibitory effect on the growth of the freshwater green algae Selenastrum capricornutum (currently known as Raphidocelis subcapitata).

The 72-hour ErC50 value for inhibition of growth rate was 32.2 µg a.s./L based on mean measured concentration. The 72-hour ErC10 for inhibition of growth rate resulted in 9.05 µg a.s./L (mean measured).

Executive summary:

The summary below is taken from the CLH report (Dec 2017) of Mancozeb:

 

A study on the toxicity of Dithane M-45 (82.4 % mancozeb) to the algal species Selenastrum capricornutum (currently known as Raphidocelis subcapitata) was performed to ‘US.EPA 1984 and ASTM 1984 standard procedures for the acute toxicity testing of freshwater green algae’, and in compliance with GLP principals. Exposure to the test item was in static conditions for 120 h at nominal formulation concentrations of 0 (control), 0.033, 0.065, 0.13, 0.25, and 0.5 mg a.s./L.

Measurements of the test substance were made throughout the study and ranged from 65 - 75 % of the nominal at 0 h, and 1.3 - 7.3 % of the nominal at 120 h. The initial measured EC50 used for the risk assessment was 0.044 mg a.s./L. The initial measured NOEC was 0.023 mg a.s./L. Although, the study was originally considered not valid during the EU Annex I renewal of Mancozeb, the endpoints quoted above were used in the risk assessment in addendum 1 to the DAR. The RMS determined that it was possible to calculate a valid (mean measured) endpoint from this study (according to the current EFSA opinion; EFSA technical Report, 2015. Ch. 3.1). The applicant provided these values which were calculated to be: ErC50 = 32.2 μg a.s./L (mm), ErC10 = 9.05 μg a.s./L (mm), EyC50 = 8.79 μg a.s./L (mm), and EyC10 = 3.54 μg a.s./L (mm). These values were considered acceptable for use in the risk assessment for the AIR 3 renewal of Mancozeb as a pesticide and will also be used for hazard classification.

Description of key information

The toxicity of the test item to the freshwater green algae Selenastrum capricornutum (currently known as Raphidocelis subcapitata) was determined in a test according US.EPA 1984 and ASTM 1984 standard procedures for the acute toxicity testing of freshwater green algae, and in compliance with GLP principals.

 

The test determined an 120-hour ErC50 of 32.2 µg a.s./L and an 120-hour ErC10 of 9.05 µg a.s./L based on mean measured concentration.

Key value for chemical safety assessment

EC50 for freshwater algae:

32.2 µg/L

EC10 or NOEC for freshwater algae:

9.05 µg/L

Additional information

The toxicity of the active substance Mancozeb (purity > 90%) as well as of the preparation Pennozep 80 WP (Mancozeb: 82.0%) and Dithane M-45 (Mancozeb: 82.4%) to algae and cyanobacteria was assessed in several guideline studies with different test species. In addition, results on toxicity to phytoplankton from an aquatic mesocosm study are available. All algal growth inhibition studies were considered unacceptable for use in the risk assessment in Addendum 1 to the DAR due to a lack of verification of the test concentrations. Nevertheless, the most sensitive endpoint (from the Forbis study (1990), Preparation, Dithane M-45, Mancozeb: 82.4%) was used in the risk assessment of the DAR in the absence of a valid algal endpoint and is considered as key information to comply with the hazard assessment applied under Regulation (EU) No 1107/2009 concerning the placing of plant protection products on the market and repealing.

 

Key study

Acute toxicity to green algae (Selenastrum capricornutum) in a static system (ref. Forbis, 1990, Doc. No. 823-019, cross reference to Risk Assessment Report according to Regulation (EU) No 1107/2009: 8.2.6/02)

The summary below is taken from the CLH report (Dec 2017) of Mancozeb:

A study on the toxicity of Dithane M-45 (82.4% mancozeb) to the algal species Selenastrum capricornutum (currently known as Raphidocelis subcapitata) was performed to ‘US.EPA 1984 and ASTM 1984 standard procedures for the acute toxicity testing of freshwater green algae’, and in compliance with GLP principals. Exposure to the test item was in static conditions for 120h at nominal formulation concentrations of 0 (control), 0.033, 0.065, 0.13, 0.25, and 0.5 mg a.s./L. Measurements of the test substance were made throughout the study and ranged from 65-75% of the nominal at 0h, and 1.3-7.3% of the nominal at 120h. The initial measured EC50 used for the risk assessment was 0.044 mg a.s./L. The initial measured NOEC was 0.023 mg a.s./L. Although the study was originally considered not valid during the EU Annex I renewal of mancozeb, the endpoints quoted above were used in the risk assessment in addendum 1 to the DAR. The RMS determined that it was possible to calculate a valid (mean measured) endpoint from this study (according to the current EFSA opinion; EFSA technical Report, 2015. Ch. 3.1). The applicant provided these values which were calculated to be: ErC50 = 32.2 μg a.s./L (mm), ErC10 = 9.05 μg a.s./L (mm), EyC50 = 8.79 μg a.s./L (mm), and EyC10 = 3.54 μg a.s./L (mm). These values were considered acceptable for use in the risk assessment for the AIR 3 renewal of mancozeb as a pesticide and will also be used for hazard classification.

 

Supporting information

The toxicity of Mancozeb to algae was evaluated in an aquatic outdoor mesocosm study by Memmert (1999). This study was re-assessed by van Wijngaard (2013) and is considered to be reliable with restrictions (Klimisch 2). The focus of the semi-field was on sediment organisms and planktonic organisms. Effect concentrations were derived based on population dynamic. 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. As a result, a consistent 77-day NOEC for Scenedsmus spp. of 400 µg Penncozeb 80 WP /L was determined. Further, a NOEAEC of < 10 μg a.s./L (< 12.5 μg/L Penncozeb 80 WP) was derived for this study. On the basis of the applied effect evaluation procedure, a NOEC_population of 3.2 μg a.s./L was determined. The NOEC_community was 10 μg a.s./L. However, the NOEC for the populational and community level were triggered by results for the zooplankton community level, not by results for phytoplankton.

 

Conclusion

A broad data basis on toxicity of Mancozeb to aquatic algae is available. Based on the most reliable information (Forbis, 1990) the ErC50 and ErC10 of Mancozeb is considered to be 32.2 μg a.s./L (mm) and 9.05 μg a.s./L (mm), respectively. The derived ErC10 is supported by 77-day NOEC for Scenedsmus spp. of 400 µg Penncozeb 80 WP/L and the conservative overall NOEAEC of < 10 μg a.s./L determined for the mesocosm study by Memmert (1999) as re-assessed by van Wijngaard (2013). The provided information is regarded reliable and sufficient to cover the data requirements according to REACH Annex VII, section 9.1.2.