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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 December 2002 to 22 May 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: samples were collected from each new batch of test solution on days 0 and 1 of the test to determine test material concentrations. Samples were also collected from the 24 hour old solution in each replicate test chamber on day 1 of the test prior to renewal and at test termination. Additional samples were collected on day 2 from the newly prepared batches of test solution prior to renewal and from the 24 hour old solutions in each replicate test chamber and stored for later analysis if required.
- Sampling method: decanting solution from mixing vessels or pipetting of solutions from mid-depth in each test chamber.
- Sample storage conditions before analysis: none
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test solutions prepared daily at nominal concentrations by added calculated amount of test material into dilution water and mixing for 22-24 hours
- Evidence of undissolved material (e.g. precipitate, surface film, etc): the test material appeared on the surface at test initiation in 12 mg/L solution
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Feeding during test: none (and two days prior to the start of the test)

ACCLIMATION
- Acclimation period: at least 14 days
- Acclimation conditions (same as test or not): yes
- Feeding frequency: daily
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
131 mg/L as CaCO3
Test temperature:
11.4 - 13.0 °C
pH:
8.1 - 8.5
Dissolved oxygen:
5.8 - 9.9 mg/L
Nominal and measured concentrations:
0.38, 0.75, 1.5, 3.0, 6.0 and 12 mg/L (nominal)

0.31, 0.62, 1.3, 2.6, 5.5 and 9.4 mg/L (actual)
Details on test conditions:
TEST SYSTEM
- Test vessel: 4 L glass jars with Teflon-lined lids
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: minimal headspace
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well on the Wildlife International site
- Metals: Mg 11.6 ppm; K 5.45 ppm; all others <5.1 ppm
- Pesticides: <250 ppb
- Alkalinity: 180 mg/L as CaCO3
- Conductivity: 310 µmhos/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light/8 hours dark
- Light intensity: 149 lux
Reference substance (positive control):
no
Key result
Duration:
96
Dose descriptor:
LC50
Effect conc.:
7.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % C.I. 5.5-9.4 mg/L
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
behaviour
Details on results:
Daily observations of mortality and other signs of toxicity are detailed in Table 1.

All fish in the negative control group appeared healthy and normal throughout the test. All fish in the 0.31, 0.62, 1.3 and 2.6 mg/L treatment groups also appeared normal throughout the test with no mortalities or overt signs of toxicity observed.

While no mortalities occurred among fish in the 5.5 mg/L treatment, sublethal effects were noted throughout the test.

Table 1: Cumulative mortality and clinical observations

   3.5 h  24 h  48 h  72 h  96 h   
Measured conc. (mg/L) Dead Effects Dead Effects Dead Effects Dead Effects Dead Effects % cumulative mortality
 0  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 0  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 0.31  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 0.31  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 0.62  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 0.62  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 1.3  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 1.3  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 2.6  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 2.6  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0/10  10AN  0
 5.5  0/10 7R; 1E; 1N; 1C  0/10  10R  0/10  10R  0/10 9R; 1N  0/10 9R; 1N  0
 5.5  0/10 6R; 3E; 1N  0/10 8R; 1A; 1E  0/10 9R; 1A  0/10 7R; 3A  0/10 9R; 1N  0
 9.4  0/10  10R  10/10  -  10/10  -  10/10  -  10/10  -  100
 9.4  0/10  10R  10/10  -  10/10  -  10/10  -  10/10  -  100

AN = appear normal

R = lying on the bottom with little motion other than gill movement

E = erratic swimming

N = loss of equilibrium

C = lethargy

A = surfacing for an unusually long period

Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the test, the 96 hour LC50 was 7.2 mg/L (95 % C.I. 5.5-9.4 mg/L). The 96 hour no-mortality concentration was 5.5 mg/L and the NOEC was 2.6 mg/L.
Executive summary:

In a GLP compliant acute fish toxicity study conducted in line with standardised guidelines OECD 203, EU Method C.1 and EPA OPPTS 850.1075, the acute toxicity to fish of the test material was determined.

Rainbow trout were exposed to the test material at nominal test concentrations of 0.38, 0.75, 1.5, 3.0, 6.0 and 12 mg/L (equivalent to measured test concentrations of 0.31, 0.62, 1.3, 2.6, 5.5 and 9.4 mg/L respectively).

Under the conditions of the test, the 96 hour LC50 of the test material was determined to be 7.2 mg/L. The 96 hour no-mortality concentration was 5.5 mg/L and the NOEC was 2.6 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 May 1995 to 21 December 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: EPA Guideline No. 72-3(a)
Deviations:
yes
Remarks:
(The test temperature slightly exceeded the range of 22 ± 1ºC, the dissolved oxygen concentration fell below 60%. These deviations did not affect the outcome or the validity of the study)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Prior to test initiation, water samples were collected from all test chambers to verify proper diluter function. 4 mL water samples were collected from the controls and all five test solutions at test initiation (day 0) and termination (day 4) to verify actual test concentrations. Water samples were collected from midway in the water column in the test chambers
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 40.4858 and 4.0491 g of test material were weighed into 500 and 50 mL volumetric flasks respectively and brining to volume with dimethylformamide. 784 µL if stock solution was injected in the chemical mixing box (total volume 3137 mL) during each diluter cycle providing the highest nominal test concentration of 20 mg/L. The test solution was proportionally diluted in the system to provide the four lower test concentrations.
- Controls: dilution water control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISM
- Common name: sheepshead minnow
- Length at study initiation (length definition, mean, range and SD): 3.2-4.5 cm (mean 3.8 ± 0.4 cm)
- Weight at study initiation (mean and range, SD): 0.82-2.28 g (mean 1.54 ± 0.42 g)
- Feeding during test: no

ACCLIMATION
- Acclimation period: 8 days
- Acclimation conditions (same as test or not): yes
- Type and amount of food: frozen brine shrimp and flake food
- Health during acclimation (any mortality observed): no diseases observed or treated
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
21.9 - 23.7 ºC
pH:
8.0-8.2
Dissolved oxygen:
4.7-7.5 mg/L (initial)
3.1-5.7 mg/L (during test)
Salinity:
20 %
Nominal and measured concentrations:
2.59, 4.32, 7.20, 12 and 20 mg/L (nominal)

2.07, 3.53, 6.44, 10.4 and 16.6 mg/L (actual)
Details on test conditions:
TEST SYSTEM
- Test vessel: 23.6 L glass tanks
- Material, size, headspace, fill volume: fill volume - 15.3 L and water height of 13 cm; test solution volume - 1400 ± 140 mL
- Type of flow-through: modified proportional vacuum-siphon diluter system
- Renewal rate of test solution (frequency/flow rate): average rate of 4.6 cycles/hour providing 10.1 volume additions every 24 hours
- No. of organisms per vessel: 20

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural saltwater pumped from a shallow saltwater well
- Metals: magnesium 742 mg/L; all others <0.01 mg/L
- Pesticides: <1.2 µg/L
- Chloride: 10,800 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light/8 hours dark
- Light intensity: 4.9-6.7 microEinsteins per square meter per second

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality (daily); abnormalities in behaviour or physical appearance (daily)

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.01, 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes (0 % mortality at ≤10 mg/L; 100 % mortality at 100 mg/L)
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
12.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % C.I. 10.4-16.6 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.53 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Daily observations of mortality and other signs of toxicity can be found in Table 1.

Table 1: Cumulative mortality and clinical observations

  24 hours    48 hours    72 hours    96 hours     
Mean measured conc. (mg/L) Dead Effects Dead Effects Dead Effects Dead Effects Cumulative % mortality
 0  0/20  20AN  0/20  20AN  0/20  20AN  0/20  20AN  0
 0 (solvent control)  0/20  20AN  0/20  20AN  0/20  20AN  0/20  20AN  0
 2.07  0/20  20AN  0/20  20AN  0/20  20AN  0/20  20AN  0
 3.53  0/20  20AN  0/20  20AN  0/20  20AN  0/20  20AN  0
 6.44  0/20 9AN; 11A  0/20 3AN; 17A  0/20 19AN; 1A  0/20 19AN; 1A  0
 10.4  1.20 1AN; 18A  1/20 17A;8N  1/20 19N; 19C  1/20 19N; 19C  5
 16.6  14/20 6N  18/20  2N  202/20  -  20/20  -  100

AN = appear normal

N = loss of equilibrium

C = lethargy

A = at the surface

Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the test, the 96 hour LC50 was 12.7 mg/L (95 % C.I. 10.4-16.6 mg/L). The 96 hour NOEC was 3.53 mg/L.
Executive summary:

In a GLP compliant acute fish toxicity study conducted in line with standardised guidelines OECD 203, EU Method C.1 and EPA Guideline No. 1 72 -3(a), the acute toxicity to fish of the test material was determined. Sheepshead minnow were exposed to the test material at nominal test concentrations of 2.59, 4.32, 7.20, 12 and 20 mg/L (equivalent to measured test concentrations of 2.07, 3.53, 6.44, 10.4 and 16.6 mg/L respectively).

Under the conditions of the study, the 96 hour LC50 of the test material was determined to be 12.7 mg/L. The 96 hour NOEC was 3.53 mg/L.

Description of key information

The LC50 of the test material, to Rainbow trout, was determined to be 7.2 mg/L and the NOEC to be 2.6 mg/L according to a study performed in line with OECD Guideline 203, EU Method C.1 and EPA OPP 850.1075.

The LC50 of the test material, to Sheephead minnow, was determined to be 12.7 mg/L and the NOEC to be 3.53 mg/L according to a study performed in line with OECD 203, EU Method C.1 and EPA Guideline No. 1 72 -3(a).

Key value for chemical safety assessment

LC50 for freshwater fish:
7.2 mg/L

Additional information

Oncorhynchus mykiss and Cyprinidon variegatus were similar in sensitivity to the test material with 96 hour LC50 values of 7.2 mg/L and 12.7 mg/L, respectively. In addition, to these two GLP studies, a review has been reported on the acute toxicity of the test material. For the test material, the 96 hour LC50 values (excluding ‘greater than values' which appear to represent outliers) ranged from 4.2 to 42 mg/L. Based on the review by Gijswijt (1980), three out of 56 endpoints, representing 24 species of fish, were lower but very similar to the endpoint for O. mykiss in the GLP study; these were 6.5, 4.2 and 6.4 mg/L. As the results from the C. variegatus study were obtained from only one replicate, the O. mykiss study is considered to be the key study for this endpoint.