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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 - 18 December 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: Dilution water control, solvent control (dimethylformamide 0.1 mg/L) and a nominal concentration of 100 mg/L of the test substance
- Sampling method: Analytical samples taken at 0 and 96 hours and analysed by HPLC-UV instrumentation
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: This study was run with a dilution water control, solvent control (dimethylformamide 0.1 mg/L) and a nominal exposure concentration of 100 mg/L of test substance as received. Four 10 L aliquots of dilution water in glass stock jars were each spiked using 1.0 mL of a 1000 g/L solution of the test substance in dimethylformamide. The contents of each stock jar were then stirred at a nominal temperature of 30°C for approximately 2 days. After stirring the stock jars were transferred to the test lab (at a nominal 15°C) until required for use (approximately 17 hours). On Day 0 of the study 40 L of dilution water were filtered under vacuum, through Whatman filter paper and then placed into each of 2 vessels (dilution water control and solvent control). A nominal 4.0 g of dimethylformamide was added to the solvent control vessel and stirred using a glass pipette to ensure good mixing. The contents of each stock jar containing test solution (40 L in total) were also filtered prior to addition to the nominal 100 mg/L test vessel.

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- Common name: rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Houghton Springs Fish Farm, Winterbourne Houghton, Blandford Forum, Dorset, DT11 0PD on 30 July 2009
- Length at study initiation (length definition, mean, range and SD): 46 - 67 mm with a mean of 58 mm
- Weight at study initiation (mean and range, SD): 1.21 – 4.47 g with a mean of 3.25 g
- Maintenance of the brood fish: Following their arrival on site the fish were held in 20% seawater for 11 days and given a twice daily prophylactic treatment of Formalin (25 ppm) and Malachite green (1 ml/100 l) for 6 days. The treatment was completed on 10 August 2009 (126 days before the start of the test) and the fish acclimated back to 100% freshwater. Fish were held in a 340 L fibreglass tank under artificial lighting and were contained in water as described for the test dilution water. Food was withheld from the fish for at least 24 hours prior to commencement of the test.

- Acclimation period: Prior to the test the fish were acclimatised for a minimum period of 7 days.
- Acclimation conditions (same as test or not): test temperature (15 ± 1°C)
- Type and amount of food during acclimation: high protein pelleted fish food
- Feeding frequency during acclimation: daily
- Health during acclimation (any mortality observed): There were no mortalities recorded within the 7 days prior to the start of the study.

- no feeding during test
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
59.0 mg/L CaCO3
Test temperature:
from 14.8 to 15.2°C
The temperatures of the dilution water in the holding tanks were monitored daily
pH values ranged from 7.3 to 7.9
Dissolved oxygen:
8.4 to 9.8 mg/l (83 to 97% of the air-saturation value)
At no time during the course of the study was the DO concentration in any of the test vessels less than 60% of the air-saturation value (6.04 mg/L)
The salinity of the dilution water control was measured prior to the start of the study and was shown to be 0.2‰
230 μS cm-1 @ 25°C.
Nominal and measured concentrations:
The mean measured concentrations of the test concentrations were calculated using the arithmetic mean of the triplicate values. On Day 0 the mean measured concentration was 88% of the nominal value and after 96 hours it was also 88%, showing no loss over the course of the study.

- Dilution water control: measured conc.: 0 hr: < 1.8 mg/L; 96 hr: < 1.8 mg/L
- solvent control: measured conc.: 0 hr: < 1.8 mg/L; 96 hr: < 1.8 mg/L
- 100 mg/L: measured conc.: 0 hr: 88 mg/L*; 96 hr: 88 mg/L*

* Results are mean of triplicate analysis and calculated as the arithmetic mean of the 0 and 96 hr resutls.
Details on test conditions:
- Test vessel: glass vessels
- Material, size, headspace, fill volume: Single glass vessels (external dimensions; 460 mm × 305 mm × 385 mm; length × width × height) were used for the dilution water control, solvent control and the exposure solution. The vessels had a working volume of 40 L.
- Aeration: gentle aeration
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): not reported
- No. of vessels per control (replicates): not reported
- Biomass loading rate: 0.81 g fish/L

- Source/preparation of dilution water: the dilution water was dechlorinated tap water, which has been passed through activated carbon, coarsely filtered to remove particulate material and dechlorinated with sodium thiosulphate. Salts were added to maintain minimum hardness levels and the treated water was then passed through an ultaviolet steriliser to a set of 20 and 10 µm filters. The supply was then delivered to a temperature controlled header tank in the test laboratory set to the nominal test temperature ± 1°C.
- Ammoniacal Nitrogen as N: < 0.002 mg/L
- Solid Susptended at 105°C: < 3.00 mg/L
- Dissolved Organic Carbon: 0.45 mg/L
- COD: < 12 mg/L
- Organochlorine pesticides: < 0.007 µg/L
- Organophosphorus pesticides: < 0.01 µg/L
- PCBs: < 0.001 µg/L
- Chlorine (as Cl2): free residual: < 2.0 µg/L; combined residual: < 2.0 µg/L (conducted on day 0)
- Alkalinity: 32.6 mg CaCO3/L
- Hardness: 59 mg/L as CaCO3
- Culture medium different from test medium: no

Final nominal concentrations in the media:
- NaHCO3: 64,8 mg/L
- CaCl2. 2H2O: 293.8 mg/L
- MgSO4.7H2O: 123.3 mg/L
- KCl: 5.8 mg/L
- Na2SiO3.5H2O: 0.747 mg/L
- NaNO3: 0.02274 mg/L
- KH2PO4: 0.0143 mg/L
- K2HPO4: 0.0184 mg/L

- Photoperiod: 16 hours fluorescent light and 8 hours dark with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered to be dead when no reaction was observed after touching the caudal peduncle and visible breathing movements were absent.

- Spacing factor for test concentrations: none (limit test)
- Range finding study: yes
Prior to the study, range finding work was undertaken to establish the concentration(s) required for the definitive study. Three fish were exposed to concentrations of the test substance at 1.0, 10 and 100 mg/L, including a solvent control, as described for the definitive
study. Although one death was seen (33%) in the 100 mg/L test concentration, this death was observed to be due to the aggressive behaviour of one dominant fish rather than a toxic effect of the test substance. It was therefore decided to conduct a limit test at a single test substance
concentration of 100 mg/l and a solvent control for the definitive study.
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
Key result
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
96 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- 24h > 100 mg/L
- 48h > 100 mg/L
- 72h > 100 mg/L
- Mortality of control: none
- Other adverse effects control: no
- Abnormal responses: General symptoms noted in this study were surfacing, sounding, loss of balance and dark discolouration. These symptoms were thought to be due to the aggressive nature of one fish in each of the affected tanks. During the test a single fish in the 100 mg/l exposure concentration became very agressive towards the remaining fish. One, smaller fish was so badly damaged that it had to be removed and humanely sacrificed while the remainder were displaying quite severe symptoms. The agressive fish was sectioned off for a number of hours during which time the health of the remaining fish improved considerably showing that the observations made were as a result of the agressive fish rather than a toxic effect.

At the end of the exposure period, the fish from the dilution water control were weighed and measured. The range was 1.21-4.47 g with a mean of 3.25 g, and the range in length was 46-67 mm with a mean of 68 mm. The loading of fish in the dilution water control was apporx. 0.81 g/L.
Validity criteria fulfilled:
The acute toxicity of the test substance was determined in a 96 hour static limit test according to the OECD guideline 203. Under the conditions of the present study, The 96-h LC50 exceeded the nominal concentration of 100 mg/L. The results of the test can be considered reliable without restriction.

Description of key information

96 hour LC50 (Oncorhynchus mykiss) > 100 mg/l (nominal). No effects observed.

Key value for chemical safety assessment

Additional information

Acute toxicity (96 hour) to fish has been investigated in the freshwater species Oncorhynchus mykiss following OECD Guideline 203 at concentrations up to 100 mg/l. The 96 hour LC50 was reported as > 100 mg/l (nominal). The 96 hour NOEC was reported as 100 mg/l (nominal).