Ziram

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

8 February 2006 - 18 July 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

a solvent was used for the study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from one test chamber of each treatment and control group two days to test initiation to confirm the operation of the diluter. Water samples were collected from alternating replicate test chambers of each treatment and control group on days 0, 7, 14, 21, 28 and 33 (test termination) to determine concentrations of the test substance in the test chambers. Sampling from the 750 and 375 µg/l tretament groups was discontinued after day 7 and day 28, respectively, due to 100 % mortality in each treatment group. All samples were collected at mid-depth in the test chambers, placed in glass vials and processed immediately for analysis.

Vehicle:

yes

Details on test solutions:

Initial stock solutions were prepared combining an appropriate amount of 14C ziram stock solution prepared in DMF with non radiolabelled ziram stock solution.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: fathead minnow
- Strain: pimephales promelas
- Source: Chesapeake Cultures, Inc. Hayes, Virginia, USA

POST-HATCH FEEDING
- Type/source of feed: live brine shrimp nauplii
- Frequency of feeding: 3 times per day during the firts seven days post-hatch. Thereafter they were fed 3 times per day on weekdays and 2 times per day per day on weekends.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Post exposure observation period:

28 d

Hardness:

132 - 140 mg/L as CaCO3

Test temperature:

24.0 - 25.0 C

pH:

8.0 - 8.1

Dissolved oxygen:

6.4 - 8.3 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0.0 (control and solvent control), 47, 94, 188, 375 and 750 µg a.i./L
Measured concentrations: 0.0, 48, 101, 195, 393 and 750 µg a.i./L

Details on test conditions:

TEST SYSTEM
- Emybro cups: glass cylinders, approx. 50 mm diameter, with 425 µm nylon scree attached to the bottom with silicone selant.
- Test vessel:
- 9-L glass aquaria filled with approx. 7 L of test solution
- Type of flow-through: continuous-flow diluter
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: 7.5 L/10 animals (post-hatch period)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: fresh water from a well approx. 40 meters deep located on the Wildlife International, Ltd. site.
_ Hardness: 132 - 144 mg CaCO3/L
- Alkalinity: 182 - 186 mg CaCO3/L
- Specifc conductance: 340 - 360 µmhos/cm


OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h darkness
- Light intensity: Fluorescent light bulbs (Colortone® 50)


EFFECT PARAMETERS MEASURED:
- day 1: embryos were observed twice for mortality and eggs with fungus.
- > day 1 until hatching was complete: observations of embryo mortality and the removal of dead embryos were performed once daily.
- Hatching > 90 % in the negative control group on day 5 of the test, the larvae were released to their respective test chambers and the post-hatch period began.
- During the 28-day post hatch exposure period, the larvae were observed daily to evaluate the number of mortalities and the number of individuals exhibiting clinical signs of toxicity or abnormal behaviour.
From these observations, time to hatch, hatching success and post-hatch growth and survival were evaluated. Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatched survival was calculated as the number of larvae surviving to test termination divided by the total of embryos hatched successfully.
Post-hatch growth of the fathead minnows was evaluated at the conclusion of the 28-day post-hatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet dry weights were measured to the nearest 0.1 mg using an analytical balance. Fish were placed in an oven at approximately 60 ºC for two days to obtain dry weight data.

Reference substance (positive control):

no

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

101 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages:
- Days to hatch : all viable embryos in the treatment replicates hatched between days 3 and 5 of the test and larvae were released into the test chambers on day 5. There were no no apparent differences in time to hatch between the control group and any of the treatment groups
- Larval survival in the 48, 101 and 195 µg/L treatment groups was 95, 97 and 72 %, respectively. No larvae survived 28 days bpost-hatch in the 383 and 750 µg/L tretament groups.
- The majority of the fish in the 48, 101 and 195 µg/L treatment groups appeared normal throughout the test. There were a few sporadic observations of organisms that appeared smaller, weak or lethargic, were swimming erratically, or had a curved spine. These observations were few in number and did not occur in a concentration-responsive pattern. Observations of weak and/or smaller fish were observed in the 393 and 750 µg/L treatment groups prior to 100 % mortality.
- Observations on body length and weight: There was a slight statistically significant decrease in total length and dry weight in the 101 µg/L treatment group in comparison to the pooled controls (Bonferroni t-test, p ≤ 0.05). However, there were no statistically significant differences in growth at the next higher concentration of 195 µg/L when compared to the controls (Bonferroni t-test, p > 0.05). The small differences from the controls in the 101 µg/L treatment group were not dose-responsive, and were not considered to be biologically meaningful.

Validity criteria fulfilled:

yes

Executive summary:

Results and discussion: All viable embryos in the control and treatment replicates hatched between days 3 and 5 of the test and larvae were released into the test chambers on day 5.

Larval survival in the 48, 101 and 195 µg/L treatment groups was 95, 97 and 72 %, respectively. No larvae survived 28 days post-hatch in the 393 and 750 µg/L treatment groups. There was a statistically significant decrease in survival in the 195, 393 and 750 µg/L treatment groups in comparison to the pooled controls (Fisher’s Exact test, p ≤ 0.05).The LOEC for larval survival was 195µg/L and the NOEC was 101 µg/L.

In the 48, 101, 195, 393 and 750µg/L treatment groups the hatching success was 98, 99, 95, 96 and 10 %, respectively. Fisher´s Exact test indicated that the decrease in hatching success in the 750 µg/L treatment group was statistically significant when compared to the pooled controls (p ≤ 0.05). Consequently, the LOEC for hatching success was 750 µg/L and the NOEC was 393 µg/L.

Concerning the growth parameter, the Bonferroni t-test indicated that there was a slight, yet statistically significant decrease in total length and dry weight in the 101µg/L treatment group in comparison to the pooled controls. However, there were no statistically significant differences in growth at the next higher concentration, 195 µg/L, when compared to the controls (p > 0.05). The small differences from the controls in the 101 µg/L treatment group were not dose-responsive, and were not considered to be biologically meaningful. Therefore, the NOEC for growth was 195 µg/L. The calculated MATC was 140 µg/L.

Water quality parameters were within acceptable limits throughout the test.

Reference 2

Endpoint:

adult fish: sub(lethal) effects

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

6 June 2001 - 6 November 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: OECD No. 215

Deviations:

yes

Remarks:

modified to mimic more realistic conditions: the test item was applied fourfold to a water-sediment system with intervals of seven days between the applications. The test was performed in a static manner.

Principles of method if other than guideline:

The modification is more realistic for plant protection products

GLP compliance:

yes (incl. QA statement)

Remarks:

The Swiss GLP Monitoring Authorities

Analytical monitoring:

yes

Details on test solutions:

- A concentrated stock solution of nominal 720 mg/L ZIRAM 76 WG (= 544 mg/L Ziram) was freshly prepared by homogeneously dispersing the test item in test water by intensive stirring for 10 minutes. This stock solution was used for the dosage of the two highest test concentrations (250 and 800 ug/L) by adding 63 and 200 mL stock solution to the 180 L test medium. For the dosage of the three lower test concentrations (8, 25 and 80 ug/L) the stock solution was diluted with test water to 144 ug/L ZIRAM 76 WG (= 109 mg/L Ziram) (60 mL stock solution were filled up to 300 mL). From this diluted stock solution 10, 32 and 100 mL were dosed to the 180 L test medium.
- The test item was applied into the water at the start of the study and in weekly intervals on Days 7, 14 and 21, respectively. The day of the first application of the test item was defined as Day 0 of the study.

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

- The fish species used in this study was bluegill sunfish (Lepomis macrochirus). The test fish were obtained from OSAGE Catfisheries Inc., Osage Beach, Missouri, USA. lmmediately after arriving at RCC the test fish were prophylacticly treated for diseases with Oxytetracycline hydrochlodride for three days. Thereafter the fish were held in local tap water (drinking water quality) for more than three weeks in the RCC laboratories without further treatment for disease. During this period the mortality rate in the test fish batch was lower than 1%. Then the test fish were acclimated to the test water in the water-sediment systems and to the test temperature and test conditions for 15 days prior to test start. During this acclimatization period the mortality rate in the test fish batch was lower than 4%, and all fish were healthy. Also during this time they were not treated for disease.
At the start of the acclimation period the body length and body wet weight of a subsample of ten randomly selected fish from the test flsh batch were measured. The mean body length of the fish was 4.2 cm, the mean body wet weight 1.03 g. Then, fish from the test fish batch with a suitable body wet weight were pre-selected, and randomly distributed to the test tanks, thirteen fish per water-sediment system.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Test temperature:

22.2 - 22.7°C (mean)

pH:

7.9-8.2

Dissolved oxygen:

> 60% air saturation

Nominal and measured concentrations:

Nominal: 8, 25, 80, 250 and 800 ug Ziram 76 WG /L
Nominal active ingredient: 6, 19, 61, 189 and 605 ug a.i./L
Measured: 92 to 106% nominal values

Details on test conditions:

- The water-sediment systems were prepared in large tanks in a temperature-controlled laboratory. The inner size of the tanks was 97 cm (Iength) x 45.5 cm (wide) x 54 cm (height). One tank with a water volume of 180 liters was used per test concentration and control. The tanks contained a sediment layer of approximately 4 cm and a water column of 40 cm height. Thus, the ratio of water column to sediment depth was 10:1 to avoid an unrealistic bw water/sediment ratio.
- Test water: Reconstituted water: analytical grade salts were dissolved in deionized water to obtain the appropriate concentrations. The test water was aerated until oxygen saturation was reached.
- Test sediment: An artificial sediment was used in the water-sediment systems. The sediment was prepared on basis of dry weights as follows:
Sphagnum peat: 5% (air dried, very finely ground to 2 mm)
Kaolin clay: 20%
Sand (Sihelco 36): 75%
Calcium carbonate (CaCO3): 0.5%
TOC = 2.3% (dw)
- The water-sediment systems were prepared six days for clearing of turbidity and conditioning before inserting the fish. Then 13 fish of the test fish batch were randomly placed into each tank tor acclimation of the test fish to the test conditions in the water-sediment systems.
At the start of the test the number of test fish were reduced to ten per test tank
- The water in this static test was freshly prepared at the setup of the water-sediment systems. Thereafter the test media were not renewed during the whole study period. The test media were continuously aerated during the test with compressed air and a glass pipette

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

336 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

189 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Validity criteria fulfilled:

yes

Reference 3

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Apr. 2007 - 11 Jan. 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

a solvent was used in the study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1500 (Fish Life Cycle Toxicity)

Version / remarks:

1996 (draft)

Qualifier:

according to guideline

Guideline:

other: EPA 540/9-86-137 (Standard Evaluation Procedure, Fish Life-Cycle Toxicity Tests)

Version / remarks:

1986

Qualifier:

according to guideline

Guideline:

other: EPA-600/8-81-011 (User’s Guide for Conducting Life-Cycle Chronic Toxicity Tests with Fathead Minnows (Pimephales promelas))

Version / remarks:

1981

GLP compliance:

yes

Remarks:

laboratories in the USA are not certified by any governmental agency, but are subject to official inspections

Analytical monitoring:

yes

Remarks:

Liquid scintillation counting

Details on sampling:

- Concentrations: Water samples were collected from one alternating replicate test chamber of each treatment and control group. Additional samples were collected as needed to confirm concentrations when a delivery or sampling error occurred, and when the high concentration treatment group was terminated. A sample of the 2000 mg/L stock solution was analyzed on Day 154 to confirm the concentration being delivered to the diluter, due to the low recovery in the test chamber on that day.
- Sampling method: All samples were collected at mid-depth from the test chambers.
- Sample storage conditions before analysis: No storage, samples were measured immediately.

Vehicle:

yes

Remarks:

dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Three stock solutions of radiolabelled test substance were prepared in DMF at nominal concentrations of 130 or 100 mg/L. The radioactivity was confirmed prior to use in the study by liquid scintillation counting (LSC). The analyses resulted in mean measured concentrations of 137, 97.5 and 107 mg/L, corresponding to 105, 98 and 107% of the expected radioactivity. Stock solutions of non-radiolabelled test item were prepared periodically during the study by dissolving non-radiolabelled test substance in DMF at a nominal concentration of 10.0 mg/mL. The stock was sonicated to aid in solubilization of the material and appeared slightly cloudy beige. Dispensing stock solutions at nominal concentrations of 250, 500, 1000, 2000 and 4000 mg/L were prepared in DMF using calculated volumes of the radiolabelled and non-radiolabelled stock solutions to achieve a nominal radioactivity of 2.5 x 105 dpm/mL in each dispensing stock. The five dispensing stock solutions were injected into the diluter mixing chambers (at a rate of 20 µL/minute during the parental-generation exposure and 10 µL/minute during the second-generation exposure) where they were mixed with well water (at a rate of 200 mL/minute during the parental-generation exposure and 100 mL/minute during the second-generation exposure to achieve the targeted test concentrations.
- Differential loading: yes
- Controls: negative control (well water) and solvent control
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium: 0.1 mL/L
- Evidence of undissolved material: All test solutions appeared clear and colorless in the test chambers at test initiation and termination. A white precipitate was present in the diluter mixing chamber for the 400 µg/L treatment group during the parental-generation exposure, although no precipitate was observed in the test chambers.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead Minnow
- Source: Chesapeake Cultures, Inc. Hayes, Virginia 23072
- Age at study initiation: embryos (<24 hr old at initiation of parental generation exposure)
- Length at study initiation: not mentioned in study. For more details on length at other timepoints refer to attachment "Rep.No.602A-106A_Attachment 4_Length and Weight of fish".
- Weight at study initiation: not mentioned in study. For more details on weight at other timepoints refer to attachment "Rep.No.602A-106A_Attachment 4_Length and Weight of fish".
- Feeding during test : Yes. The first feeding began when greater than 90% of the negative control individuals hatched in the parental generation, or when greater than 90% hatch occurred in each second-generation replicate.
- Food type: Newly hatched larvae were fed newly-hatched brine shrimp nauplii (Artemia sp.). Parental-generation fish were fed brine shrimp nauplii or frozen brine shrimp (after 29 days post-hatch commercial flake food was fed additionally). Second-generation fish were fed brine shrimp nauplii or frozen brine shrimp.
- Amount: Rations were adjusted proportionally each week, if necessary, based on the number of fish in each test chamber and the developmental stage of the fish.
- Frequency: Newly hatched larvae were fed three times per day during the first seven days post-hatch. Parental-generation fish were fed brine shrimp nauplii or frozen brine shrimp three times daily on weekdays and at least twice daily on weekends and holidays. Second-generation fish were fed three times daily.
- Other: Fish were not fed during either the final 48 hours prior to growth measurements at the end of the adult exposure period or at the end of the 28-day growth period of the second-generation to allow the fish to clear their digestive tracts before weight measurements were made.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

303 d

Remarks on exposure duration:

The first generation exposure lasted 275 d and the second generation test lasted additional 28 d.

Hardness:

Parental generation experiment: 124 - 148 mg/L as CaCO3
Second generation experiment: 128 - 148 mg/L as CaCO3

Test temperature:

Parental generation experiment: 24.5 - 25.9 °C
Second generation experiment: 24.7 - 25.6 °C

pH:

Parental generation experiment: 8.0 - 8.3
Second generation experiment: 8.2 - 8.3

Dissolved oxygen:

Parental generation experiment: 5.8 - 8.4 mg/L
Second generation experiment: 6.9 - 8.4 mg/L

Conductivity:

Parental generation experiment: 320 - 365 µmhos/cm
Second generation experiment: 320 - 380 µmhos/cm

Nominal and measured concentrations:

Nominal concentrations: negative control, solvent control, 25, 50, 100, 200 and 400 µg/L
Mean measured concentrations during parental generation test: Mean measured concentrations during second generation test:

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria
- Size of vessel, material, fill volume:
- Parental generation (embryo/larvae/juvenile, Day 0-61) and 2nd generation (embryo/larvae/juvenile, 28-d post-hatch): 9 L glass aquaria filled with 7 L of test solution (glass cylinders approximately 50 mm in diameter with 425-um nylon screen attached to the bottom with silicone sealant)
- Adult exposure (Day 61-275): 54-L glass aquaria filled with approximately 27 L of test solution were used. During spawning period (Days 182 - 275), each of the two adult test chambers was divided into two spawning compartments (30 x 24 x 15 cm) using stainless steel dividers. The remaining space in the tanks was used as a third compartment to hold auxiliary fish for reproduction.
- Type of flow-through: A continuous-flow diluter was used to deliver each concentration of the test substance and the controls. Syringe pumps (Harvard Apparatus) were used to deliver the five test substance stock solutions and dimethyl formamide (DMF) for the solvent control into mixing chambers indiscriminately assigned to each treatment and the control. The flow of dilution water to the mixing chambers was controlled by rotameters. The flow of test water from each mixing chamber was split and allowed to flow into the replicate test chambers.
- Renewal rate of test solution:
- Parental generation: 10 volume additions per day in each of four replicate test chambers per treatment and control group.
- Adult exposure: 5 volume additions per day in each of two replicate test chambers per treatment and control group.
- No. of organisms per vessel: 50 embryo thined to 25 larvae, thined again to 25 juvenile fish and reduced to 12 after Day 177 post-hatch when fish reached sexual maturity.
- No. of vessels per concentration (replicates): 4 during embryo and larvae exposure, 2 during juvenile exposure
- No. of vessels per control (replicates): 4 during embryo and larvae exposure, 2 during juvenile exposure
- No. of vessels per vehicle control (replicates): 4 during embryo and larvae exposure, 2 during juvenile exposure
- Biomass loading rate: 0.39 g of fish per liter of test solution
- Instantaneous loading (total wet weight of fish per liter of water in the tank): 2.10 g of fish per liter of test solution
- Other: Periodically during the test, the organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The freshwater used for culturing and testing was obtained from a well approximately 40 meters deep located on the laboratory site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to delivery to the diluter system, the water again was filtered (0.45 µm) to remove fine particles, and then passed through an ultraviolet (UV) sterilizer.
- Chloride: 12.8 mg/L
- Alkalinity: 178 – 184 mg/L as CaCO3
- Ca/mg ratio: 34.9/12.8 mg/L
- Conductivity: 290-300 µmhos/cm
- Culture medium different from test medium: no
- Pesticides: please refer to attachment "Rep.No.602A-106A_Attachment 3_Analyses of Pesticides, Organics and Metals"


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:
- Parental generation: photoperiod adjusted at approximately two-week intervals to approximate the photoperiod of Evansville, Indiana (Day length 10:30 - 15:45 h, 30-minute transition period)
- Second generation: 16 h light / 8 h darkness (30-minute transition period)
- Light intensity: 107 - 572 Lux

EFFECT PARAMETERS MEASURED:
- Growth (parental and second generation): Data included total photographic length of parental-generation juvenile fish at Days 28 and 56 post-hatch, wet weight of parental-generation fish thinned at 56 days post-hatch, total length and wet weight of adult fish thinned on Day 177 post-hatch and at test termination, as well as total length and wet and dry weight of second-generation juvenile fish at Day 28 post-hatch.
- Hatching success: Data on time to hatch was evaluated by visual interpretation of the data.
- Survival (parental and second generation): Data included viability of parental and second-generation embryos through the period of hatching (hatching success) and survival of the parental and second-generation fry during the growth period.
- Reproduction (parental generation): Data included spawning frequency (the number of days that eggs were found on spawning substrates for each reproductive group) and the total number of eggs deposited for each reproductive group.

Key result

Dose descriptor:

NOEC

Effect conc.:

24 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth

Remarks on result:

other: parental generation

Dose descriptor:

NOEC

Effect conc.:

424 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatching success

Remarks on result:

other: parental generation

Dose descriptor:

NOEC

Effect conc.:

51 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival

Remarks on result:

other: parental generation

Dose descriptor:

NOEC

Effect conc.:

51 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: parental generation

Dose descriptor:

NOEC

Effect conc.:

202 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatching success

Remarks on result:

other: second generation

Dose descriptor:

NOEC

Effect conc.:

202 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival

Remarks on result:

other: second generation

Dose descriptor:

NOEC

Effect conc.:

202 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth

Remarks on result:

other: second generation

Details on results:

- Observed effects at each concentration for each observation time: please refer to attachment "Rep.No.602A-106A_Attachment 2_Biological Results"
- Concentrations that produce lethal or other effects: please refer to attachment "Rep.No.602A-106A_Attachment 2_Biological Results"
- Cumulative mortality at each concentration and for each recommended observation time if possible: please refer to attachment "Rep.No.602A-106A_Attachment 2_Biological Results"
- Mortality in the controls: please refer to attachment "Rep.No.602A-106A_Attachment 2_Biological Results"
- Clinical observation of the fish: please refer to attachment "Rep.No.602A-106A_Attachment 5_Clinical Observations"
- Effect concentrations exceeding solubility of substance in test medium: please refer to attachment "Rep.No.602A-106A_Attachment 1_Analytical data"

The measured concentrations in the test chambers at test start (parental and second generation test) ranged from approximately 94 to 108% of nominal concentrations.

The measured concentrations in samples collected during the parental generation exposure ranged from approximately 81 to 118% of nominal concentrations. When the measured concentrations of the samples collected during the parental-generation were averaged for each treatment group, the mean measured concentrations ranged from 96 to 106% of nominal concentrations.
The measured concentrations in samples collected during the second generation exposure ranged from approximately 95 to 116% of nominal concentrations. When the measured concentrations of the samples collected during the second-generation were averaged for each treatment group, the mean measured concentrations ranged from 101 to 106 of nominal concentrations.

The highest concentration treatment group (400 µg/L) was not continued into the second-generation exposure.

Evaluating effects on reproduction in adult fish was confounded by effects on growth and development, which made it difficult to form reproductive groups in the affected treatment groups.

Table 1: Validity Criteria of OPPTS 850.1400.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The dissolved oxygen concentration should be >60% of the air saturation value throughout the test.	dissolved oxygen was always > 60%	yes
The water temperature should not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species.	Max difference of temperature was 1.4°C	yes
Evidence must be available to demonstrate that the concentrations of the test substance in solution have been satisfactorily maintained within ± 20 percent of the mean measured values.	Measured concentrations were always > 80% of nominal values	yes
Overall survival of fertilized eggs in the controls and, where relevant, in the solvent-only controls must be greater than or equal to the limits defined in Tables 4. and 5. under paragraphs (h)(1)(ii) and (h)(1)(iii) of this guideline.	Hatching success was > 90%	yes
When a solubilizing agent is used it must have no significant effect on survival nor produce any other adverse effects on the early-life stages as revealed by a solvent-only control.	No significant differences compared to control (water) – controls were therefore pooled	yes

Validity criteria fulfilled:

yes

Remarks:

For details please refer to field "any other information on results incl. tables"

Description of key information

NOEC (270 d) = 24 µg/L (geom. mean meas., based on growth of Pimephales promelas, EPA OPPTS 850.1500)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

24 µg/L

Additional information

Three studies are available assessing the long-term toxicity of zinc bis dimethyldithiocarbamate (CAS No. 137-30-4) to fish.

In the key study (2008) Pimephales promelas were exposed to the substance over a full life cycle to evaluate effects on hatch, survival, growth and reproduction. The study was conducted according to the EPA Guidance OPPTS 850.1500 and GLP. In a flow-through system mean measured concentrations ranged from 24 to 424 µg/L during the parental-generation exposure and from 26 to 202 µg/L during the second-generation exposure. A radiolabeled test item was used and was analytically monitored via liquid scintillation counting. The first generation exposure lasted 275 d and the second generation exposure lasted additional 28 d. The NOECs during the parental-generation exposure for hatching success, survival, growth and reproduction were 424, 51, 24 and 51 µg/L (mean measured), respectively. The NOECs during the second-generation exposure for hatching success, survival and growth were 202, 202 and 202 µg/L (mean measured), respectively. The most sensitive endpoint measured was growth in the parental generation, which was chosen as key value for the environmental assessment of this endpoint.

The chronic toxicity of zinc bis dimethyldithiocarbamate (CAS No. 137-30-4) to Pimephales promelas was investigated in a supporting study (2006) according to OECD Guideline No. 210, EPA Guideline OPPTS 850.1400 and GLP. Fish embryos were exposed for 33 days to the test substance within a flow-through water regime. Hatching success was significantly reduced at the highest tested concentration (750 µg/L) whereas growth of the surviving larvae at the end of the test did not differ between treatment groups and controls. Larval mortality reached 100% at concentrations of 393 and 750 µg/L and was significantly reduced at the 195 µg/L concentration. Thus, the NOEC (33 d) was determined to be 101 µg/L.

An additional supporting study (2001) was conducted according to OECD Guideline No. 215, with modifications (inclusion of sediment) in order to mimic realistic conditions in the environment. This test was performed under GLP conditions. The formulation Ziram 76 WG was used as test substance. Lepomis macrochirus were exposed to the test substance in a water/sediment system for 28 days, with a four-fold application regime (application of test item on days 0, 7, 14 and 21 of the study). The resulting NOEC (28 d) was 189 µg a.i. /L.

Furthermore, the effects of the substance on the early life stages of the marine species Sheepshead Minnow (Cyprinodon variegatus), were investigated under flow-through conditions for a period of 34 days (6 days hatching period and 28 days post hatch). The study (2006) was conducted according to the EPA Guideline OPPTS 850.1400 and GLP. Fish were exposed to nominal concentrations of 28, 56, 133, 225 and 450 µg/L as well as to a negative control and a solvent control (corresponding to mean measured concentrations of <LOQ, <LOQ, 27, 58, 117, 222 and 443 µg/L). Due to statistical significant reduced larval survival and growth at the treatment level of 443 µg/L, the NOEC and the LOEC based on larval growth and survival were determined to be 222 and 443 µg /L respectively.