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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 1987 to 12 September 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: 12.5 mL taken from each vessel and composited to a total volume of 50 mL on each sample day (with adjustments made where mortality was seen). 10 mL of toluene was added to each sample and the samples shaken vigorously for 3 minutes. The toluene phase was then removed, diluted with further toluene if necessary and analysed. Samples were taken on days 0, 1, 7 and 14, every 7 days thereafter until day 96.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Chemical name of vehicle: acetone
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Eggs fertilised at testing laboratory prior to test initiation

POST-HATCH FEEDING
- Start date: day 50
- Type/source of feed: live brine shrimp nauplii (with additional commercial fish food as the fish became larger)
- Frequency of feeding: twice daily (increased during test on basis of average fish size)
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 d
Hardness:
272-278 mg/L
Test temperature:
9-12 °C
pH:
7.9-8.3
Dissolved oxygen:
6.9-9.9 mg/L
Nominal and measured concentrations:
0.312, 0.625, 1.25, 2.5 and 5 mg/L (nominal)

0.33, 0.66, 1.2, 2.4 and 5.1 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Emybro cups: 9.5 cm diameter x 14 cm high glass tubing with screening silicone glued to the bottom. Cups were oscillated vertically to ensure water exchange.
- Test vessel: glass aquaria
- Material, size, headspace, fill volume: 15.7 x 305 cm with a water depth of 24 cm yielding an approximate 11.5 litre replicate chamber
- Aeration: yes
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate): 6.9 times in a 24 hour period
- No. of fertilized eggs/embryos per vessel: 30 (test solutions); 50 (controls)
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water

OTHER TEST CONDITIONS
- Photoperiod: 16 hour light/8 hour dark after the embryos had hatched into fry
- Light intensity: 59.6 ± 15 foot candles

EFFECT PARAMETERS MEASURED: embryo viability (after 11 day exposure); survival for fry at day 34 and 60 post-hatch; abnormal (sublethal) or physical changes and mortality daily; growth of fry on days 70 and 96

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 2.0 and 4.0 mg/L
- Results used to determine the conditions for the definitive study: yes

POST-HATCH DETAILS
- Begin of post-hatch period: day 36
- No. of hatched eggs (alevins)/treatment released to the test chamber: fifteen sac-fry after thinning on day 46
- Release of alevins from incubation cups to test chamber on day no.: 46
Reference substance (positive control):
no
Key result
Duration:
96 d
Dose descriptor:
other: Maximum Acceptable Toxicant Concentration
Effect conc.:
0.79 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Details on results:
Viability of the newly fertilised eggs (day 11) used for the initiation of this study averaged 96 % for the control.

Hatchability of rainbow trout eggs after 41 days of continuous exposure to the test material was significantly affected in the highest test concentration of 5.1 mg/L, when compared to the control.

Survival of fish at day 34 and day 60 post-hatch was significantly reduced in the highest test concentration of 5.1 mg/L, when compared to the control.

Growth, as measured by standard length, was significantly reduced after 34 days of exposure in all test concentrations. However, this effect was transient at lower concentrations as the reduction in length does not appear in the two lowest test concentrations at 60 days post-hatch.

Growth of fish, as measured by wet weight after 60 days of exposure, was significantly reduced in the three highest test concentrations.

An overview of the mean results can be found in Table 1.
Reported statistics and error estimates:
Comparison analyses between water, solvent controls and test levels were performed using the parameters hatchability, survival, standard length and wet weight. The statisitcal data were analysed by Systat(R).
One-way analysis of variance (ANOVA) calcualtions were used to determine if there were any significant differences between the controls and the treatment groups. If there were no significant differences, individual replicate data were composited by concentration and analysed by one-way analysis of variance using data from the control. If treatment effects were indicated following a significant F-test of the mean square ratios, Tukey's HSD multiple means comparison test was used to determine which exposure levels differed from the control values.

Table 1: Mean percentage hatch, survival, standard lengths and wet weights of rainbow trout

   34 days post-hatch  60 days post-hatch 
Mean measured conc. (mg/L) Mean egg hatch (%) Survival (%) Mean standard length (mm) Survival (%) Mean standard length (mm) Mean weight (mg)
 Control  75  95  29 ± 2.8  93  40 ± 3.4  956 ± 217
 Solvent control  66  93  29 ± 2.3  93  40 ± 2.6  974 ± 217
 0.33 ± 0.031  73  72  27* ± 2.8  72  39 ± 3.2  926 ± 210
 0.66 ± 0.061  73  88  26* ± 2.5  85  39 ± 2.5  887 ± 180
 1.2 ± 0.12  57  80  24* ± 3.6  73  36* ± 3.1  711 ± 166
 2.4 ± 0.16  63  88  24* ± 2.2  88  34* ± 2.5  584* ± 147
 5.1 ± 0.45  35  17*  17* ± 2.4  15*  21* ± 1.6  100* ± 2.6

*significantly different from control values (P<0.05)

Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the test, the Maximum Acceptable Toxicant Concentration (MATC) limits for the test material were estimated to be the mean measured concentrations of 0.66 mg/L and 1.2 mg/L based on survial and growth data. The MATC, defined as the geometric mean of the LOEC and the NOEC, was 0.79 mg/L.
Executive summary:

In a GLP compliant early life-stage fish toxicity study conducted in line with standardised guideline EPA Guideline 72-4, the toxicity to ealry-life stage fish of the test material was determined. Rainbow trout were exposed to the test material at nominal test concentrations of 0.312, 0.625, 1.25, 2.5 and 5 mg/L (equivalent to measured test concentrations of 0.33, 0.66, 1.2, 2.4 and 5.1 mg/L respectively). Under the conditions of the test, the Maximum Acceptable Toxicant Concetration was determined to be 0.79 mg/L.

Description of key information

The Maximum Acceptable Toxicant Concentration of the test material was determined to be 0.79 mg/L according to a study performed in line with EPA Guideline 72-4 (equivalent to a NOEC of 0.56 mg/L).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.56 mg/L

Additional information

In a GLP compliant early life-stage fish toxicity study conducted in line with standardised guideline EPA Guideline 72-4, the toxicity to early-life stage fish of the test material was determined. Rainbow trout were exposed to the test material at nominal test concentrations of 0.312, 0.625, 1.25, 2.5 and 5 mg/L (equivalent to measured test concentrations of 0.33, 0.66, 1.2, 2.4 and 5.1 mg/L respectively). Under the conditions of the test, the Maximum Acceptable Toxicant Concentration was determined to be 0.79 mg/L (equivalent to a NOEC of 0.56 mg/L in line with Table R.10 -1 of 'Guidance on information requirements and chemical safety assessment Chapter R.10').