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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12th January 2012 to 14th August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1500 (Fish Life Cycle Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Primary stock solutions of Mancozeb were generally prepared fresh at each sampling event in 10 % Na2EDTA in HPLC grade water at concentrations ranging from 0.577 to 0.783 mg a.s./mL. Subsequent dilutions were made in 10 % Na2EDTA in HPLC grade water to prepare matrix spiking solutions and analytical standard solutions. One of these subsequent dilutions was further diluted in dilution water and put through the hydrolysis reaction. Following the hydrolysis reaction, analytical standards were made by subsequent dilutions of the CS2-iso-octane-extract and were used for the analytical confirmation of the definitive test samples. All solutions were stored at room temperature when not in use.

The concentrations of Mancozeb in test solutions were determined in samples collected nine days prior to initiation (Study Day -9) of the definitive test, Study Day 0 (test initiation), and at least weekly thereafter including Study Day 215 (test termination). A single replicate sample was collected from the control and test-substance treatments; alternating replicates were sampled at each sampling event. The concentrations of Mancozeb in the diluter stock solutions were determined in samples collected on the same days as the test solutions.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Diluter stock solutions were prepared approximately every one to four days from 29 November 2011 to 13 August. Diluter stock solutions were prepared approximately every one to four days from 29 November 2011 to 13 August 2012.

Appropriate amounts of Mancozeb, corrected for active ingredient, were dissolved in appropriate volumes of dilution water for a concentration of 4.0 mg a.s./L. Each stock solution was prepared in a 5-L glass carboy containing a silicon stopper, a Teflon stir bar, and a glass pipet that was inserted through the stopper down to the bottom of the water column. ABC reagent water added to the carboys and the dissolved oxygen was depleted by sparging the water for at least one hour by bubbling nitrogen (N2) gas into the carboys prior to adding the test substance. ABC reagent water is produced by passing reverse-osmosis water through a series of deionization tanks, a laboratory water purification system consisting of carbon, de-mineralization, and organic adsorption cartridges, and then through a 0.2-µm filter. Approximately 60 mL of N2 sparged water was drawn from the carboy and used to transfer/rinse the pre-weighed test substance from the holding container to the carboy. The mixture was again allowed to sparge with N2 gas for at least 15 minutes after the test substance addition, then the carboy was sonicated in an ultrasonic waterbath for approximately one hour. The carboy was placed on a magnetic stir plate and contents stirred while covered with dark opaque plastic and N2 sparging. After at least seven hours of stirring, the carboy was placed on the diluter system. Stirring and N2 sparging continued while on the diluter system. All materials that contacted the test solutions were glass or PTFE.
The FMI metering pump introduced appropriate volumes of the diluter stock solution to the chemical mixing box, where the delivered volume was diluted with approximately 4,100 mL of dilution water. Mancozeb stock solution usage was monitored and recorded daily. All diluter stock solutions were stored at room temperature, shielded from light.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Strain: not reported
- Source: In-house culture

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: eggs were gently rolled off the spwaing substrate with a gentle circular motion of a gloved finger into a glass dish and visually assessd for fertilization under magnification
- Subsequent handling of eggs: number of fertilized eggs versus total number of eggs was used to calculate the percent fertilization

POST-HATCH FEEDING
F0-fish:
Start: Study day 4
- Type/source of feed: live brine shrimp nauplii (plus standard commercial food on study day 65)
- Amount given: ad libitum
- Frequency of feeding: three times daily

F1-fish:
- Type/source of feed: live brine shrimp nauplii (plus standard commercial food at approx. 16 days after hatch)
- Amount given: ad libitum
- Frequency of feeding: three times daily

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

215 d

Hardness:

130 to 176 mg CaCO3/L (F0 growth/spawing chambers)
142 to 152 mg CaCO3/L (F1 growth chambers)

Test temperature:

24.2 °C - 25.4 °C (F0 growth/spawing chambers)
24.5 °C - 25.2 °C (F1 growth chambers)

pH:

7.8 to 8.8 (F0 growth/spawing chambers)
7.8 to 8.5 (F1 growth chambers)

Dissolved oxygen:

4.0 to 9.3 mg/L (51 to 118% saturation) (F0 growth/spawing chambers)
2.7 to 8.3 mg/L (34 to 100% saturation) (F1 growth chambers)

Nominal and measured concentrations:

Nominal: 0 (control), 0.50, 1.0, 2.0, 4.0 and 8.0 µg/L
Measured conc: 0 (control), 0.382, 0.694, 1.35, 2.58, and 5.05 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: full glass aquaria
- Material, size, headspace, fill volume: glass, for F0 spawning units: 22 cm wide x 38 cm, long x 21.5 cm high, depth of 12 cm, test solution volume 10 L; F0 growth and F1 test chambers: 15.3 cm wide x 21.7 cm long x 24.2 cm high, solution depth of 14.4 cm, test solution volume of 5 L.
- Type of flow-through: 2-L proportional diluter system with electromagnetic dosing pump
- Renewal rate of test solution (frequency/flow rate): at least six volumes of test solution in a 24-h period
- No. of fertilized eggs/embryos per vessel: 50 embryos
- No. of vessels per concentration: 4
- No. of vessels per control: 4

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark
- Light intensity: 317 to 820 lux

POST-HATCH DETAILS
- Begin of post-hatch period: Study Day 5
- No. of hatched eggs (alevins)/treatment released to the test chamber: 25 fries
- Release of alevins from incubation cups to test chamber on day no.: Study day 8 (day 3 post-hatch)

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 25

Duration:

215 d

Dose descriptor:

EC10

Effect conc.:

1.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

cumulative number of eggs

Key result

Duration:

215 d

Dose descriptor:

EC10

Effect conc.:

1.27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

number of eggs/F0-female/day

Duration:

215 d

Dose descriptor:

NOEC

Effect conc.:

1.35 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Cum. No. eggs

Duration:

215 d

Dose descriptor:

NOEC

Effect conc.:

2.58 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number of spawns

Key result

Duration:

215 d

Dose descriptor:

NOEC

Effect conc.:

1.35 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

No of eggs/F0-female/day

Details on results:

Please refer to section "Any other information on results incl. tables" and the attached document.

Reported statistics and error estimates:

The no-observed-effect concentration (NOEC) for egg hatchability and fish survival data were determined by using a Fisher’s exact test to analyse number hatched or number of surviving organisms. A Hochberg adjustment was used to control the experiment-wise error rate for the Fisher’s test at the same alpha level. In addition, the NOEC for these parameters was estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett’s test with the alternate hypothesis being the mean proportion hatched/surviving differed from the control mean. Prior to the Dunnett’s test, a Shapiro-Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance, respectively, of the raw and transformed values over treatments. When the results from the Shapiro-Wilk’s and Levene’s tests indicated normality (p > 0.01) and homogeneity of variance (p > 0.01), a parametric analysis was performed using the non-transformed data. When the results from the Shapiro-Wilk’s and Levene’s tests indicated non-normality (p < 0.01) and/or unequal treatment variances (p < 0.01), a nonparametric analysis was performed on the ranks of the data.
The day of hatch start and end, growth and reproduction were evaluated using an ANOVA procedure and a two-tailed Dunnett’s test with the alternate hypothesis being the mean for the parameter was different than the control mean. Prior to the Dunnett’s test, a Shapiro-Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance, respectively, of the raw and transformed values over treatments.

Following result summary was taken from the RAR of Macozeb (Vol. 3 CA, B9, 2018):

Analytical measurements

Table 1: Summary of analytical results from the definitive test

Target Nominal Concentration (μg a.s./L)	N (number of measurements used in mean calculation)	Mean Measured Concentration (μg a.s./L)	Mean Measured Conc. Percent of Nominal	Mean Measured Conc. Percent of Day 0 Measured Conc.
0 (control)	27	< MQL	NA	NA
0.50	30	0.382	76	108
1.0	35	0.694	69	99
2.0	41	1.35	68	107
4.0	36	2.58	65	102
8.0	41	5.05	63	89
MQL: Minimum quantifiable limit; NA: not applicable

The overall mean measured concentrations of mancozeb during the definitive exposure in the 0.50, 1.0, 2.0, 4.0, and 8.0 μg a.s./L nominal treatments were 0.382, 0.694, 1.35, 2.58, and 5.05 μg a.s./L, respectively, which represented recoveries of 63 to 76% of nominal treatment concentrations and 89 to 108% of Day 0 measured concentrations.

 

Effect Results

Hatchability, survival and growth

The hatchability of fathead minnow F0–embryos exposed to mancozeb was not adversely affected in any test substance treatment as compared to the control. Post-hatch survival of the F0-minnows was not adversely affected in any test substance treatment on Study Days 65, 118-119, and 152 as compared to the control. There was a statistically significant effect of mancozeb concentration on survival at 5.05 μg a.s./L on the spawning group- study day 167 (one-tailed Dunnett’s test; p= 0.0154).

The standard length of the F0-minnows on Study Day 65 was not significantly increased or reduced in any test substance treatment as compared to the control. The body weight (i.e., blotted wet weight) and standard length of the F0-female minnows on Study Days 118-119, 152 and 167 were not significantly increased or reduced in any test substance treatment as compared to the control. The body weight and standard length of the F0-male minnows on study days 118-119 was statistically significantly less than the control (one-tailed Dunnett’s test; p= 0.0334). The body weight and standard length of the F0-male minnows on Study Days 152 and 167 were not significantly increased or reduced in any test substance treatment as compared to the control.

The days to first spawn by F0-female minnows was not adversely affected in any of the test substance treatments as compared to the control. The number of spawns by the F0-female minnows and the percent of fertile eggs produced by the F0-female minnows was statistically significantly reduced in the 5.05 μg a.s./L treatment (two-tailed Dunnett’s test; p= 0.0010, two-tailed Dunnett’s test; p= 0.0234) . The number of eggs per F0-female minnow per day and the cumulative number of eggs produced by the F0-female minnows was statistically significantly reduced in the 2.58 and 5.05 μg a.s./L treatments (two-tailed Dunnett’s test; p≤ 0.0019, two-tailed Dunnett’s test; p≤ 0.0017 respectively). The most sensitive endpoints were number of eggs per F0-female minnow per day and the cumulative number of eggs produced by the F0-female minnows.

 

Sublethal Effects

No sub-lethal effects were noted in the F0 Spawning Group (study days 120-167) in mancozeb concentrations of 2.58 μg a.s./L or below. As there was then an increase to 32 observed sub-lethal effects at 5.05 μg a.s./L, this was considered a significant, treatment-related effect.

Because of the fluctuating nature of sub-lethal observations throughout treatment groups and study days, all other sub-lethal observations did not exhibit a clear dose-effect relationship with treatment. The nature, magnitude and severity of the sublethal effects indicated reproduction remained as the most sensitive endpoint for this exposure.

 

Reproduction effects

Table 2: Summary of ECx results based on mean measured concentrations

Endpoint	EC10 (μg a.s./L)	95% CL	EC20 (μg a.s./L)	95% CL	EC50 (μg a.s./L)	95% CL	Statistically significant conc./ reseponse found?
Cumulative number of eggs (162 dph)	1.30	0.036-1.928	1.65	0.149-2.289	2.61	1.509-4.730	Yes
No. eggs/female/day (162 dph)	1.27	0.047-1.906	1.63	0.177-2.275	2.61	1.536-4.652	Yes
Percent fertility (162 dph)	3.12	n.d.	4.07	n.d.	6.79	n.d.	No
Number of spawns (162 dph)	2.17	n.d.	2.88	n.d.	4.95	n.d.	No
Male length (114 dph)	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	Yes
Male fresh weight (114 dph)	2.49	n.d.	4.69	n.d.	n.d.	n.d.	Yes
Male length (147 dph)	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	No
Male fresh weight (147 dph)	3.12	n.d.	4.43	n.d.	8.66	n.d.	No
CL: Confidence limit, dph: days post hatch, n.d.: not determined

 

The ECx values for the endpoints: Hatch F0, F1; Survival F0 60, 114, 147, 162 dph; Survival F1 before and post reduction; Length and Weight F0 60 and 162 dph (males and females); Length and Weight F1 56 dph (males and females); female Length and Weight 114, 147 dph; F0 Spinal curvature; and F1 Days to first spawn were not calculated since LOEC was at or above the highest test concentrations (5.05 μg a.s./L) and there was no clear concentration/response relationship observed (according to expert judgement). The lowest NOEC calculated from this study was 1.35 μg a.s./L (mm) based on the parameters ‘number of eggs per female per day’ and ‘cumulative number of eggs’. The lowest EC10 was also calculated from the parameter ‘number of eggs per female per day’ (1.27 μg a.s./L). The EC10 calculated for the parameter ‘cumulative number of eggs’ was only slightly higher than this (1.30 μg a.s./L). The lower of these EC10 values will therefore be considered in the context of the risk assessment.

 

Validity criteria fulfilled:

yes

Conclusions:

As sub-lethal effects were observed (in most cases) across all treatment groups, including the control, it was concluded that mancozeb had no effect on the development of sub-lethal traits (except for spinal curvature in the Days 120-152 F0 Growth Group). However, this is based on the assumption that sub-lethal effects are to be expected in normal control conditions.

Based on the most sensitive reproduction endpoint of number of eggs produced per F0-female per day a NOEC of of 1.35 μg a.s./L and an EC10 of 1.27 μg a.s./L (mean measured) derived. The EC10 of 1.27 μg a.s./L will be considered in the risk assessment.

Executive summary:

In the Fish Full Life-Cycle-Test with the fathead minnow (Pimephales promelas) under flow-through conditions following nominal concentrations were tested: 0 (control), 0.50, 1.0, 2.0, 4.0, and 8.0 μg a.s./L, which corresponds to 0 (control), 0.382, 0.694, 1.35, 2.58, and 5.05 μg a.s./L based on mean measured concentrations. Each treatment consisted of four replicate test chambers tested for F0 and F1 generations.

Following endpoint summary was taken from the RAR of Macozeb (Vol. 3 CA, B9, 2018): F0 female growth and survival was mostly unaffected by the mancozeb concentrations measured in this study, except in the cases of the non-lethal effect of spinal curvature (which is considered to be treatment-related, and therefore a significant effect) (NOEC: 2.58 μg a.s./L) and the survival of the study day 167 spawning group (NOEC: 2.58 μg a.s./L). F0 male weight and length was negatively affected by mancozeb concentration (NOEC: 2.58 μg a.s./L) on study days 118-119. F1 survival was negatively affected by mancozeb concentration (NOEC: 2.58 μg a.s./L).

Reproduction-related endpoints were the most negatively affected by mancozeb concentration of those measured in this study. The number of spawns and the percentage of fertile eggs a female produced were negatively affected by mancozeb concentration (NOEC: 2.58 μg a.s./L in both cases). The most sensitive endpoints were number of eggs per F0-female minnow per day and the cumulative number of eggs produced by the F0-female minnows (NOEC: 1.35 μg a.s./L in both cases). The corresponding EC10 values were 1.30 μg a.s./L for the endpoint cumulative number of eggs and 1.27 μg a.s./L for number of eggs per F0-femal per day. Based on these most sensitive endpoints, the overall NOEC and LOEC values were 1.35 and 2.58 μg a.s./L, respectively. The lower EC10 of 1.27 μg a.s./L will be considered in the risk assessment.

Description of key information

In a Fish Full-Life-Cycle Test according to EPA OPPTS 850.1500 with Pimephales promelas a NOEC of 1.35 µg/L (mean measured) was determined. The reproduction endpoints number of eggs per F0-female per day and the cumulative number of eggs produced by the F0-female were the most sensitive endpoints (NOEC: 1.35 μg a.s./L in both cases). The EC10 of 1.27 μg a.s./L based on the endpoint  number of eggs per F0-female per day is considered as key value for the risk assessment.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

1.27 µg/L

Marine water fish

Marine water fish

Dose descriptor:

EC10

Effect concentration:

2.88 µg/L

Additional information

The long-term toxicity of the active substance Mancozeb (purity > 90%) as well as of Dithane M-45 (Mancozeb: 78.4%) to fish was assessed in several Early Life Stage Toxicity tests with different test species and in one Full Life-Cycle test with Pimephales promelas. The Fish Full Life-Cycle test is evaluated as the most reliable study (Hicks, 2012, classified Klimish 1) and considered as key information.

Long-term toxicity to fish (Pimephales promelas) under flow-through conditions (Hicks 2012 (Doc. No. 826-008, cross reference to Risk Assessment Report according to Regulation (EU) No 1107/2009: KCA 8.2.2.2/01))

In the Fish Full Life-Cycle-Test with the fathead minnow (Pimephales promelas) under flow-through conditions following nominal concentrations were tested: 0 (control), 0.50, 1.0, 2.0, 4.0, and 8.0 μg a.s./L, which corresponds to 0 (control), 0.382, 0.694, 1.35, 2.58, and 5.05 μg a.s./L based on mean measured concentrations. Each treatment consisted of four replicate test chambers tested for F0 and F1 generations.

Following endpoint summary was taken from the RAR of Macozeb (Vol. 3 CA, B9, 2018): F0 female growth and survival was mostly unaffected by the mancozeb concentrations measured in this study, except in the cases of the non-lethal effect of spinal curvature (which is considered to be treatment-related, and therefore a significant effect) (NOEC: 2.58 μg a.s./L) and the survival of the study day 167 spawning group (NOEC: 2.58 μg a.s./L). F0 male weight and length was negatively affected by mancozeb concentration (NOEC: 2.58 μg a.s./L) on study days 118-119. F1 survival was negatively affected by mancozeb concentration (NOEC: 2.58 μg a.s./L).

Reproduction-related endpoints were the most negatively affected by mancozeb concentration of those measured in this study. The number of spawns and the percentage of fertile eggs a female produced were negatively affected by mancozeb concentration (NOEC: 2.58 μg a.s./L in both cases). The most sensitive endpoints were number of eggs per F0-female minnow per day and the cumulative number of eggs produced by the F0-female minnows (NOEC: 1.35 μg a.s./L in both cases). The corresponding EC10 values were 1.30 μg a.s./L for the endpoint cumulative number of eggs and 1.27 μg a.s./L for number of eggs per F0-femal per day.

Based on these most sensitive endpoints, the overall NOEC and LOEC values were 1.35 and 2.58 μg a.s./L, respectively. The EC10 of 1.27 μg a.s./L will be considered in the risk assessment.

The key study was peer reviewed by rapporteur member states in accordance with Regulation (EU) No 1107/2009 and has been proven to be valid by the EFSA (European Food Safety Authority (EFSA), approved 12 June 2019, doi:10.2903/ j.efsa.2019.5755).

 

Additionally, a long-term test with the salt water species Cyprinodon variegatus was conducted according to the EPA Guideline OPPTS 850.1400 under GLP conditions (Hicks, S.L., 2011, Mancozeb: Early Life-Stage Toxicity Test with the Sheepshead Minnow, Cyprinodon variegatus, Under Flow-Through Conditions; ABC Laboratories Inc. - 66202). The test item was tested at nominal concentrations of 0, 1.9, 3.8, 7.5, 15, 30, 4000 μg a.s./L which were analytically determined to be <MQL*, 0.918, 2.13, 4.46, 9.04, 19.5, N/A as mean measured concentrations. The test run for 39 days. After 29 days of post-hatch (Day 39 of the study) growth, surviving fish were euthanized and measured for standard length and blotted wet weight. Based on the obtained results the evaluating authority determined that the mean measured NOEC of 0.918 μg a.s./L is considered protective and therefore this value will be considered as reliable for the risk assessment. An EC10 on the endpoint fresh weight was determined to be 2.88 µg a.s./L and was accepted by the evaluator.