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Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 February 2006 - 18 July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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.

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

Description of key information

NOEC (33d) = 101 µg a.i./L (Pimephales promelas)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
NOEC
Effect concentration:
101 µg/L

Additional information

The chronic toxicity of zinc bis dimethyldithiocarbamate (CAS No. 137-30-4) to Pimephales promelas was investigated by Palmer et al. (2006). This test was performed according to OECD Guideline No. 210 and EPA OPPTS 850.1400, Fish, Early-Life Stage Toxicity Test (1992, 1996), under GLP conditions. 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 a.i./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 a.i./L and was significantly reduced at the 195 µg a.i./L concentration. Thus, the NOEC (33 d) was determined to be 101 µg a.i./L.

 

An additional study (Memmert, 2001) was conducted according to OECD Guideline No. 215, Fish, Juvenile Growth Test (2000) with modifications (inclusion of sediment) in order to mimic realistic conditions in the environment. This test was performed under GLP conditions. The test organisms were exposed to zinc bis dimethyldithiocarbamate 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.