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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988- 06-27 to 1988-07-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US EPA FIFRA 72.4

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for analytical confirmation were taken on day 0, 4, 7, 14 and 21 of the study. Analysis of water samples for the test item was performed as follows: duplicate 10-mL water samples were taken from replicate beakers A & B on days 0, 7 and 21 and from C & D on days 4 & 14 of each test level and control. Each sample received 10 mL of J. T. Baker Hydrocount scintillation cocktail and was analyzed by LSC. Also a 10 mL aliquot of the diluter stock solution was taken, 10 mL Hydrocount were added and the sample was analyzed by LSC.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The diluter stock solutions were prepared in the following manner, 3.0 mg of pure, dry Dithane M-45 DAP Chronic Stock Sample (specific activity 3600 DPM/µg) was placed directly on the solenoid valve system. On a timed basis, 400 mL of dilution water flushed the test compound into the mixing chambers which had 100 mL of dilution water already placed inside. The total volume of dilution water was now 500 ml. After stirring for 1 hour in the mixing chamber this solution became the diluter stock solution which had a nominal concentration of 6.0 mg/L total product or 5.0 mg a.i./L. The diluter system injected a 10 mL aliquot of this stock solution into the diluter mixing box (1000 mL).

Test system: A half-liter proportional diluter system was used for the intermittent introduction of dilution water and Dithane M-45 Into the test chambers. Since the Dithane M-45 compound hydrolyzed rapidly, a system was necessary to provide fresh stock solutions at 6-hour intervals. The apparatus constructed for this purpose consisted of a microprocessor-based programmable timer that operated a series of solenoid valves connected to four mixing chambers and a manifold assembly. Four pre-measured aliquots of pure, dry Dithane M-45 were added to the system once daily. Water was then allowed to flush the test compound into the mixing chambers on a timed basis. The test compound was allowed to stir on a magnetic stirplate for 1 hour In the mixing chambers. After stirring for 1 hour this solution became the diluter stock solution. Solenoid valves released this stock solution into the manifold assembly on a timed basis. The manifold assembly was attached to a Hamilton* Micro Lab 420 syringe dispenser which provided the stock solution to the proportional diluter system. The proportional diluter system contained seven sets of four replicate one-liter test chambers, designated as control and level 91 through level #5. Flow-splitting chambers were utilized to thoroughly mix and divide each Dithane M-45 concentration for delivery to the test chambers. To minimize turbulence, the influent water was introduced into the test chambers via 14-gauge hypodermic needles. One-liter glass beakers, labeled A, B, C and D, were used as test chambers. These chambers had notched drains which were covered with 50-mesh stainless steel
to prevent escape of the test daphnids. The test daphnids were placed in each of the quadruplicate chambers. The dilution water used In this study was a blend of R.O. (reverse osmosis) water and ABC well water. The water was blended to give a hardness of between 160-180 mg/1 as CaCO. This blended water was delivered to each test chamber at an average rate of 4.1 mL/min, an amount which was sufficient to replace the 1-Liter test volume on an average of 5.9 times in a 24-hour period. The test chambers were immersed in a temperature controlled water bath held at 20°C (± 2 °C).
- Controls: blank control
- Evidence of undissolved material: no
- Other relevant information:

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: < 24 hours old
- Stage and instar at study initiation: first instar

- Source: laboraty culture, originally obtained from Columbia National Fisheries Research Laboratory (CNFRL)
- Feeding during test: yes
- Food type: algae, Tetramin, cereal leaves, vitamins and yeast
- Amount: 2 x 10^8 algae cells total to each replicate plus 9.0 mg/mL suspension of Tetramin, cereal leaves, vitamins and yeast giving a final suspended solids concentration of 1.8 mg/L
- Frequency: three times daily with algae suspension and one time per day supplemental feeding with Tetramin, cereal leaves, vitamins and yeast

ACCLIMATION
- Acclimation period: no

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

not specified

Test temperature:

20 +/- 2 °C

pH:

7.9 – 8.3

Dissolved oxygen:

6.8 to 8.3 mg/L

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal concentrations: 3, 5.9, 12, 25 and 50 μg a.i./L; mean measured concentrations: 2.9, 7.3, 12, 26 and 53 μg a.i./L.

Details on test conditions:

TEST SYSTEM
- Aeration: no supplemental aeration during test
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate): 1 L water 6 times a day, flow rate was 4.1 mL/min
- No. of organisms per vessel: 10
- No. of vessels per concentration: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see attached table on water source and parameters

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light with 30 min dawn and dusk transition phase
- Light intensity: 50-70 foot-candles
- Light quality: white fluorescent bulbs

EFFECT PARAMETERS MEASURED: survival, abnormal effectsand occurrence of first brood daily. Growth, reproduction parameters. The primary objective of the study was to estimate the maximum acceptable toxicant concentration (MATC)
range (the lower value is the concentration that causes no observable deleterious effects for any parameters measured during the study: The upper value is the concentration that produce at least one deleterious effect on the measured parameters).

TEST CONCENTRATIONS
Two preliminary tests were conducted with non-radio-labeled Dithane M-45. The first test was a 96-hour static renewal study at the nominal concentrations of 0.10, 0.50 and 1.0 mg/L total product which corresponds to 0.082, 0.41 and 0.82 mg/L active Ingredient. After 96-hours the 1.0 mg/L (0.82 mg a.l./L) test level had 70% mortality while the 0.50 mg/L (0.41 mg a.i./L) level had 20% mortality. The 0.10 mg/L (0.082 mg a.i./L) level had no mortality, however, 90% of the daphnids were affected. A second, static, test was conducted at the nominal concentration of 0.01 mg/L (0.0082 mg a.i./L). After 72-hours no mortality or abnormal effects were observed.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

10.9 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 53 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

Statistical analysis of survival of Daphnia magna after a 21-day exposure to Dithane® M-45 indicated that daphnid survival in the 53 μg a.i./L test level was significantly different from control. The MATC limits for survival were estimated to be 26 and 53 μg a.i./L. The day 21 EC50 was estimated to be > 53 μg a.i./L. The daphnid lengths at the mean measured concentration of 53 μg a.i./L were significantly different (p<=0.05) from control. The MATC limits for length were estimated to be 26 and 53 μg a.i./L. The time in days to first brood for the control and all treatment levels was 9 days. No significant difference in time to first brood occurred in this study. The mean young/adult/reproduction day for 21 days was significantly affected in the mean measured exposure levels of 12, 26, and 53 μg a.i./L for Dithane® M-45. The estimated MATC limits for reproduction were 7.3 and 12 μg a.i./L. Therefore based on the statistical analysis of survival, mean adult length, time until first brood and young/adult/reproduction day determined for this study, the MATC limits were estimated to be mean measured concentrations of 7.3 and 12 mg a.i./L.

The results of this study were re-assessed by the Rapporteur Member State UK in accordance with Regulation (EU) No 1107/2009 as the EC10 and EC20 values must be presented along with the NOEC/EC50 where it is possible to calculate them from the experimental design. As these values were not presented in the original study summary, a request was sent to the applicant to provide these values. The results sent by the applicant are attached to the section "Attached background material".

Validity criteria fulfilled:

yes

Conclusions:

In a chronic flow-through study according to US EPA FIFRA 72.4 similar to the current OECD TG 211 with Daphnia magna, the 21-day NOEC and EC10 based cum. offspring per survived parent was determined to be 7.3 μg a.s./L (mm) and 10.9 μg a.s./L (mm), respectively.

Executive summary:

A dynamic 21-day life cycle toxicity study with Daphnia magna exposed to Dithane M-45 was conducted to determine the Maximum Acceptable Toxicant Concentration (MATC) range. A half-liter proportional diluter system was used to maintain constant test concentrations using radiolabeled Dithane M-45 to facilitate measurements of test solutions. Exposure concentrations of Dithane M-45 as Mancozeb were measured analytically on days 0, 4, 7, 14 and 21. The nominal concentrations were 3.0, 5.9, 12, 25 and 50 µg a.i./L. The mean measured concentrations, as determined by liquid scintillation counting (LSC) were 2.9, 7.3, 12, 26 and 53 µg/L. The measured levels of test substance reported in this study were within ± 20% (97 % to 124 %) of the nominal. However, the endpoints were reported in terms of mean measured concentrations. The active substance Mancozeb was measured by gas chromatography (GC) on days 0, 7 and 21. Standard water quality parameters were measured throughout the study and were within acceptable limits. Statistical analysis of survival for Daphnia magna after a 21-day exposure to Dithane M-45 indicated that daphnid survival in the 53 µg/L test level was significantly different from the control. The MATC limits for survival were estimated to be 26 and 53 µg/1. A 21-day EC50 was estimated to be > 53 µg/L. The daphnid lengths in the Dithane M-45 mean measured concentration of 53 µg/L were significantly different (p ≤ 0.05) from the control. The MATC limits for length were estimated to be 26 and 53 µg/L. The time in days to first brood for the control and all treatment levels was 9 days. No significant difference in time to first brood occurred in this study. The mean young / adult / reproduction day for 21 days was significantly affected in the mean measured exposure levels of 12, 26 and 53 µg/L for Dithane M-45. The estimated MATC limits for reproduction were 7.3 and 12 µg/L. Based on the statistical analysis of survival, mean adult length, time until first brood and young / adult / reproduction day, from this 21-day Daphnia magna dynamic life cycle study, the MATC limits were estimated to be the Dithane M-45 mean measured concentrations of 7.3 and 12 µg/L. The point MATC value was calculated to be 9.4 µg/L. The EC10 and EC20, as required by commission regulation number 283/2013, were derived by the Rapporteur Member State in the course of the risk assessment according to Regulation 1107/2009 (please refer to the RAR for Mancozeb). For cumulative offspring per introduced parent, the standard non-linear regression procedure was terminated without achieving convergence due to mathematical problems. For immobility, no statistically significant concentration / response was found. The originally concluded NOEC from this study (7.3 μg a.s./L) was based on the number of young per adult per day for 21 days. However, the calculations performed by the applicant demonstrated that the NOEC for the cumulative number of offspring per survived parent was also 7.3 μg a.s./L. The lowest EC10 was also concluded from this parameter, and will therefore be considered further in the context of the risk assessment. The EC10 for reproduction (10.9 μg a.s./L mm) is considered acceptable for use in the risk assessment.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

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 dynamics

Remarks on result:

other: NOEC derived for Cladocera (including Daphnia magna)

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 dynamics

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:

other: NOEAEC

Effect conc.:

< 10 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: population dynamics

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:

63 d

Dose descriptor:

NOEC

Effect conc.:

400 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: population dynamics

Remarks on result:

other: test species: Daphnia magna

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 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01st April 2011 - 29th April 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.33, 0.65, 1.3, 2.5, 5.0 µg a.s./L
- Sampling method: The concentrations of mancozeb in test solutions were determined in samples collected eighteen days prior to initiation (day -N) of the definitive test and on days 0 (initiation), 7, 14, 21, and 28 (termination) of the definitive test. A single replicate sample was collected from the control and test-substance treatments; alternating replicates were sampled at each sampling event (e.g., replicate C sampled on day 7; replicate D sampled on day 14, etc.). The concentrations of mancozeb in the diluter stock solutions were determined in samples collected on the same days as the test solutions.
- Sample storage conditions before analysis: not reported

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Diluter stock solutions were prepared at least every four days a target concentration of 4.0 mg a.i./L from 11 March to 25 April 2011 by diluting approximately 0.0228 g (i.e., 0.0180 g as active ingredient) of mancozeb per 4.5 L of N2 sparged water. An FMI metering pump introduced 4.8-mL volumes of the diluter stock solution to the diluter system, where the diluter stock solution volume was diluted with approximately 3,825 mL of dilution water

- Control: dilution-water control
- Evidence of undissolved material: no

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid
- Strain/clone: not reported
- Age at study initiation (mean and range, SD):
- Source: not reported
- Feeding during test: yes.
- Food type: brine shrimp nauplii (Artemia sp. <48 hours old).
- Amount: ad libitum
- Frequency: three times daily

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Test temperature:

24.2 to 25.0 °C

pH:

7.92 to 8.12

Dissolved oxygen:

6.91 to 7.22 mg/L (96 to 102% saturation)

Salinity:

18.8 to 21.9%

Nominal and measured concentrations:

nominal: 0 (control), 0.33, 0.65, 1.3, 2.5 and 5.0 µg a.s./L,
measured: 0 (control), 0.217, 0.625, 0,882, 1.64 and 3.25 µg a.s./L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria (22 cm wide, 38.5 - 77 cm long, 30 cm high, volume 12 or 24 L (test chambers could be divided in two tanks))
- Aeration: no
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate): 120 L of dilution-water control and test solution to each chamber per day while one side of the test chamber was in use. 240 of dilution-water control and test solution to each chamber per day while both sides were in use. Thus, approx. 10 volume assitions in a 24-hour period.
- No. of organisms per vessel: 30 mysids
- No. of vessels per concentration: 3
- No. of vessels per control: 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:laboratory saltwater prepared by adding a commercial sea salt mix (Crystal Sea Marinemix; Marine Enterprises International, Inc. Baltimore, Maryland) to laboratory freshwater at a target salinity 20 ± 2‰

OTHER TEST CONDITIONS
- Photoperiod: 14 hours light/ 10 hours dark
- Light intensity: 448 to 548 lux

EFFECT PARAMETERS MEASURED : mortality and sublethal effects were daily recorded (Survivial of mysids, number of young per female, mean lenght of male mysids, mean length of female mysids)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Range finding study
- Test concentrations: 0 (control), 0.1, 0.20, 0.40, 0.80, 1.6, and 5.0 μg a.s./L
- Results used to determine the conditions for the definitive study: after 21 days of exposure, the mean percent survival of the mysids was 100, 97, 100, 97, 97, 97 and 50% in the 0 (control), 0.1, 0.20, 0.40, 0.80, 1.6, and 5.0 μg a.s./L treatments, respectively

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

3.25 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: Mean total young per F0-female mysid

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

3.25 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

Length of F0 female and male mysids

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

1.71 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: F0 survival

Key result

Duration:

11 d

Dose descriptor:

NOEC

Effect conc.:

1.64 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: F1 survival

Key result

Duration:

28 h

Dose descriptor:

NOEC

Effect conc.:

1.64 µg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: F0 survival

Details on results:

- Observations on body length and weight: The 28-day mean body length for the F0-male mysids was 6.06 mm in the control and ranged from 5.87 mm in the 1.64 µg a.s./L test substance treatment to 6.08 mm in the 0.217 µg a.s./L test substance treatment. The 28-day mean body length for the F0-female mysids was 6.44 mm in the control and ranged from 6.29 mm in the 1.64 µg a.s./L test substance treatment to 6.59 mm in the 0.625 µg a.s./L test substance treatment. There was no statistically significant reduction in F0-male or female 28-day mean body lengths as compared to the control in any of the test-substance treatments. The NOEC and LOEC for 28-day F0-male and female mean body length were 3.25 and >3.25 µg a.s./L, based on the mean measured concentrations

- Other biological observations:
Brood pouches were first evident on day 10 of the exposure in the control and all test-substance treatments. The mean day of first-brood release by F0 mysids was 16.8 days in the control and ranged from 15.7 days in the 0.882 µg a.s./L test substance treatment to 17.0 days in the 3.25 µg a.s./L tests substance treatment. There was no statistically significant delay in mean day of first-brood release between the control and test-substance treatments. Based on mean measured concentrations, the NOEC and LOEC for time to release of first brood were 3.25 and >3.25 µg a.s./L, respectively.
The mean number of total young produced per female during the 28-day exposure was 26.1 young in the control and ranged from 24.5 young in the 3.25 µg a.s./L test substance treatment to 36.0 young in the 0.882 µg a.s./L test substance treatment. There was no statistically significant reduction in the number of young produced per female as compared to the control in any of the test-substance treatments. Based on nominal concentrations, the NOEC and LOEC for mean number of total young produced per female were 3.25 and >3.25 µg a.s./L, respectively. The control pairs produced an average number of young that exceeded 3 young per reproductive day (i.e., per individual brood release).
Mean survival of F1 mysids after 4 days of exposure was 100% in the control and ranged from 93% in the 3.25 µg a.s./L treatment to 100% in the 0.217, 0.882, and 1.64 µg a.s./L treatments (Table 10). After 7 days of exposure, mean survival of F1 mysids was 100% in the control and ranged from 91% in the 3.25 µg a.s./L treatment to 100% in the 0.217, 0.882, and 1.64 µg a.s./L treatments. After 11 days of exposure, mean survival of F1 mysids was 98% in the control and ranged from 89% in the 3.25 µg a.s./L treatment to 100% in the 0.217, 0.882, and 1.64 µg a.i./L treatments. There were a statistically significant reduction in F1-mysid survival rates on day 4, 7, and 11 in the 3.25 µg a.s./L test-substance treatment as compared to the control. Based on mean measured concentrations, the NOEC, LOEC, and MATC values for 4-, 7-, and 11-day survival were 1.64, 3.25, and 2.31 µg a.s./L, respectively.
The 11-day mean body length for the F1-male mysids was 4.81 mm in the control and ranged from 4.62 mm in the 3.25 µg a.s./L test-substance treatment to 5.01 mm in the 0.625 µg a.s./L test-substance treatment. The 11-day mean body length for the F1-female mysids was 4.82 mm in the control and ranged from 4.86 mm in the 1.64 µg a.s./L treatment to 5.22 mm in the 0.625 µg a.s./L treatment. There was no statistically significant reduction in F1-male or female mysid mean body length, as compared to the control in any of the test-substance treatments. Based on mean measured concentrations, the NOEC and LOEC for 11-day F1-male and female mean body length was 3.25 and >3.25 µg a.s./L, respectively.

- Mortality of control: 5%
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

All statistical analyses were performed using SAS software (SAS version 9.1 for Windows). The NOECs, based on percent survival, survival of second generation offspring, reproduction (i.e., young per female), and adult length, were estimated using a one-way analysis of variance (ANOVA) procedure and either one-tailed Fisher's test with Hochberg's familywise adjustment for significance or a one-tailed Dunnett's test. The alternate hypothesis was that the mean for the parameter in the treated exposures was reduced in comparison to the negative control mean for that same parameter. The time to first brood release was analyzed using a one-tailed Dunnett's test to determine significant enhancing effects on this parameter. For all analyses, prior to the Dunnett's test, a Shapiro-Wilk test for normality and Levene's test for homogeneity of variance over treatments were conducted at each time point. When the p values from the Shapiro-Wilk's and Levene's test were greater than 0.01, indicating normality and insignificant heterogeneity, the analysis was performed on the raw value. A log transformation, or rank if these transformations were not adequate, was used when the p value was less than 0.01. The assumptions of normality and homogeneity of variance were met for all growth (i.e., body length) and reproduction data (i.e. day to first brood and total young per female); therefore, a parametric analysis was performed on these data. The assumptions of normality and homogeneity of variance were not met for all other parameters; therefore, a nonparametric analysis was performed on the ranks of the data. The maximum acceptable toxicant concentration (MATC), was calculated as the square root of the product of the NOEC and LOEC concentrations for the most sensitive toxicological endpoint. Median lethal concentration (LC50) values and their 95% confidence limits could not be established because mortality was <50% in all treatments.

Please refer to the attached document for results on the analytical measurement and the biological effect results in tabular format.

Validity criteria fulfilled:

yes

Conclusions:

The most sensitive NOEC from this study was 1.64 µg a.s./L (mean measured) based on F0 survival on day 28, and F1 survival on days 4, 7 and 11. The lowest EC10 of 1.71 μg a.s./L (mean measured) was determined for the 28-day F0 survival. The NOEC of 1.64 μg a.s./L will be considered in the risk assessment.

Executive summary:

In the Full Life-Cycle-Test with the saltwater mysid (Americamysis bahia) under flow-through conditions following nominal concentrations were tested: 0 (control), 0.33, 0.65, 1.3, 2.5 and 5.0 µg a.s./L, 0 (control), 0.217, 0.625, 0,882, 1.64 and 3.25 µg a.s./L based on mean measured concentrations. Each treatment consisted of three replicate test chambers tested for F0 and F1 generations.

Follwing summary is taken from the RAR of Mancozeb (Vol 3 CA, B9, 2018): The test acceptability criteria for this study were met. The water-quality characteristics remained within the tolerance limits set forth in the protocol. Survival of the control F0 mysids was 95%. The percentage of control F0-female mysids available to produce young that actually did produce a brood was 100%. The control pairs produced an average number of young that exceeded 3 young per reproductive day (i.e., per individual brood release) and the average total number of young produced per control F0-female mysid was 26.1.

Based on mean measured concentrations of Mancozeb during the 28-day exposure, the NOEC and LOEC values for F0-mysid survival on days 7, 13, 14, and 21 were 3.25 and > 3.25 µg a.s./L, respectively. The NOEC and LOEC values for all male and female mysid length endpoints were 3.25 and > 3.25 µg a.s./L, respectively, for both generations (F0 and F1). The NOEC, LOEC, and MATC values for F0 mysid survival at 28 days, and F1 mysid survival on days 4, 7, and 11 were 1.64, 3.25, and 2.31 µg a.s./L, respectively.

Description of key information

Long-term toxicity to aquatic invertebrates: freshwater species

In a chronic flow-through study according to US EPA FIFRA 72.4 similar to the current OECD TG 211 with Daphnia magna, the 21-day NOEC and EC10 based cum. offspring per survived parent was determined to be  7.3 μg a.s./L (mm) and 10.9 μg a.s./L (mm), respectively.

 

Long-term toxicity to aquatic invertebrates: marine species

In a mysid Full-Life-Cycle Test according to EPA OPPTS 850.1350 with Americamysis bahia a NOEC of 1.64 µg a.s./L (mean measured) was determined. The 28-day survival of F0 mysids and the F1 survival of days 4, 7 an 11 were the most sensitive endpoints (NOEC: 1.64 μg a.s./L in both cases). The lowest EC10 of 1.71 μg a.s./L derived based on the endpoint  F0 survival after 28-days of exposure. The NOEC of 1.64 μg a.s./L is considered as key value for the risk assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

10.9 µg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

NOEC

Effect concentration:

1.64 µg/L

Additional information

The long-term toxicity of the Mancozeb preparation Dithane M-45 (Mancozeb: 78.4%) to aquatic invertebrates was assessed in a Daphnia magna Reproduction tests (Burgess, 1988) and in one Full Life-Cycle test with Americamysis bahia (Hicks, 2011). The Reproduction test with Daphnia magna (classified Klimish 1) is considered as key information for freshwater species and the mysid Full Life-Cycle test (classified Klimish 1) as key information for marine species. In addition, supporting data from an freshwater mesocosm study (Memmert, 1999; re-assessed by van Wijngaard, 2013; classified Klimish 2) is available.

 

Key studies

Chronic toxicity of Dithane® M-45 to Daphnia magna under flow-through conditions (Burgess 1988 (Doc. No. 827-001, cross reference to Risk Assessment Report according to Regulation (EU) No 1107/2009: KCA 8.2.5/01))

The 28d flow-through study on Daphnia magna by Burgess (1988) with a Dithane M-45 (Mancozeb: 82.4%) had a reproduction NOEC of 7.3 μg a.s./L. Measurements of test concentration were made regularly throughout the study in all test levels. Although measured levels were within ± 20 % of the nominal, the study report states endpoints in terms of mean measured concentrations. The  RMS considers this approach acceptable for the risk assessment, and so the mean measured NOEC 7.3 μg a.s./L is considered appropriate for use in the risk  ssessment. This NOEC was based on the number of offspring per parent per day. However, upon request the applicant provided calculations of ECx values from this study. These calculations demonstrated that the NOEC for the cumulative number of offspring per surviving parent was also 7.3 μg a.s./L. The lowest EC10 calculated from the data is 10.9 μg a.s./L, also based upon cumulative number of offspring per surviving parent, and is considered acceptable for use in the risk assessment.

 

Long-term toxicity to mysid (Americamysis bahia) under flow-through conditions (Hicks 2011 (Doc. No. 829-002, cross reference to Risk Assessment Report according to Regulation (EU) No 1107/2009: KCA 8.2.5.2/01))

The summary below is partly taken from the RAR of Mancozeb (Vol. 3CA, B9, 2018): In the Full Life-Cycle-Test with the saltwater mysid (Americamysis bahia) under flow-through conditions following nominal concentrations were tested: 0 (control), 0.33, 0.65, 1.3, 2.5 and 5.0 µg a.s./L, 0 (control), 0.217, 0.625, 0,882, 1.64 and 3.25 µg a.s./L based on mean measured concentrations. The test acceptability criteria for this study were met. The water-quality characteristics remained within the tolerance limits set forth in the protocol. Survival of the control F0 mysids was 95%. The percentage of control F0-female mysids available to produce young that actually did produce a brood was 100%. The control pairs produced an average number of young that exceeded 3 young per reproductive day (i.e., per individual brood release) and the average total number of young produced per control F0-female mysid was 26.1. Based on mean measured concentrations of Mancozeb during the 28-day exposure, the NOEC and LOEC values for F0-mysid survival on days 7, 13, 14, and 21 were 3.25 and > 3.25 µg a.s./L, respectively. The NOEC and LOEC values for all male and female mysid length endpoints were 3.25 and > 3.25 µg a.s./L, respectively, for both generations (F0 and F1). The NOEC, LOEC, and MATC values for F0 mysid survival at 28 days, and F1 mysid survival on days 4, 7, and 11 were 1.64, 3.25, and 2.31 µg a.s./L, respectively.

Both key studies were peer reviewed by Rapporteur Member States in accordance with Regulation (EU) No 1107/2009 and have been proven to be valid by the EFSA (European Food Safety Authority (EFSA), approved 12 June 2019, doi:10.2903/ j.efsa.2019.5755).

 

Supporting information

Additional information on the toxicity of Mancozeb to aquatic invertebrates 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 dwelling and planktonic organisms. Effect concentrations were derived based on population dynamic, whereby only the most dominant zooplankton species (including Daphnia magna) were studied in detail. 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. In result, a consistent 63-day NOEC = 400 µg L Penncozeb 80 WP/L for Daphnia magna was determined. The 77-day NOEC for Cladocera was found to be = 10 µg a.s./L. Further, a an overall 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, an overall NOEC_population of 3.2 μg a.s./L was determined. The overall NOEC_community was 10 μg a.s./L. The NOEC for the populational and community level were triggered by results for the zooplankton community level.

 

Conclusion

In a mysid Full-Life-Cycle Test by Hicks (2011) according to EPA OPPTS 850.1350 with the marine mysid Americamysis bahia a NOEC of 1.64 µg a.s./L (mean measured) was determined. The 28-day survival of F0 mysids and the F1 survival of days 4, 7 and 11 were the most sensitive endpoints (NOEC: 1.64 μg a.s./L in both cases). The lowest EC10 of 1.71 μg a.s./L derived based on the endpoint  F0 survival after 28-days of exposure. In a chronic flow-through study according to US EPA FIFRA 72.4 similar to the current OECD TG 211 with Daphnia magna, the 21-day NOEC and EC10 based cum. offspring per survived parent was determined to be  7.3 μg a.s./L (mm) and 10.9 μg a.s./L (mm), respectively. These values are supported by results of a chronic semi-field study by Memmert (1999, re-assessed by van Wijngaard, 2013). In this higher-tier study a 63-day NOEC of 400 µg L Penncozeb 80 WP/L for Daphnia magna and a 77-day NOEC for Cladocera of 10 µg a.s./L was determined. The overall NOEAEC was found to be  < 10 μg a.s./L (< 12.5 μg/L Penncozeb 80 WP). The overall NOEC_population was 3.2 μg a.s./L and the  NOEC_community was 10 μg a.s./L. Based on the most reliable information (Hicks, 2011; Burgess, 1988) and by applying a worst case approach a NOEC of 1.64 μg a.s./L is considered as key value for marine species and the 21-day EC10 of 10.9 µg/L for freshwater species. The provided information is regarded reliable and sufficient to cover the data requirements according to REACH Annex IX, section 9.1.5.