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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-08-16 to1993-10-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
Test conducted according to the Toxic Substances Control Act (TSCA), Guideline no. EG-9 and the Support Document no. ES-6, which is similar to OECD guideline 203.
Deviations:
yes
Remarks:
higher number of replicates, fish size slightly exceeding guideline recommendation (by 0.1 to 0.2 mm).
Principles of method if other than guideline:
The final test was conducted in accordance with the Toxic Substances Control Act (TSCA) Guideline no. EG-9 and the Support Document no. ES-6. The test design is similar to the design of OECD guideline 203. The duration of the test was 96 hours. The test was carried out under a 16 h light-8 h dark regime with transition periods of ca. 30 minutes in a temperature-controlled room. The temperature in the test vessels was 22 ± 1°C. Two replicate aquaria for each concentration and the control were realised in the test. Each replicate contained 10 fish. The average standard length and weight of twenty fish of the batch used (± standard deviation) were 3.1 ± 0.1 cm and 0.46 ± 0.08 g respectively.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 3.2, 5.6, 10, 18, 32, 56 mg/L (nominal); samples from the control medium and all test solutions were taken at t = 0 h (newly prepared solutions) and t = 24 h (spent solutions after 24 h). However, the spent solution of 56 mg/L was sampled at t = 3 h, because at that time all test fish at this concentration were dead.
- Sampling method: Samples of ca. 50 mL were taken in glass bottles, filtered through a 0.45 µm filter and then transferred to the Department of Analytical Chemistry.
- Sample storage conditions before analysis: refrigerator 4 °C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The dilution water used was diluted DSWL (Dutch Standard Water Linschoten), prepared from ground water. This DSWL has proven to be suitable for keeping the test organism used in this test. At the request of the sponsor dilution water with a hardness of < 180 mg/L (expressed as CaCO3) was used and therefore DSWL was diluted with distilled water (volume ratio DSWL: distilled water = 4:1) to reach a hardness, expressed as CaCO3 of < 180 mg/L (Optimum for Pimephales promelas). : In the final test six concentrations of 2-ethylhexyl lactate were tested. Initially five concentrations were prepared. Because at t = 24 h only one fish was dead at 32 mg/L (the highest test concentration) an extra concentration of 56 mg/L was added at that time to the test concentration series.
Two portions of 200 mg of 2-ethylhexyl lactate were accurately weighed out and each portion was dissolved in two litres of dilution water, with the aid of ultrasonic vibration, giving solutions of 100 mg/L. From these solutions, two portions of each 320, 560 and 1000 mL were diluted with dilution water to ten litres to achieve test concentrations of 3.2, 5.6 and 10 mg/L. The remaining test concentrations were prepared by dissolving two portions of 180, 320 and 560 mg of the test substance in ca. three litres of dilution water with the aid of a magnetic stirrer and subsequently diluting with dilution water to ten litres to achieve test concentrations of 18,32 and 56 mg/L.
- Eluate: no
- Differential loading: no
- Controls: Diluted DSWL was used as a control medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle applied
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
- Other relevant information
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow (Pimephales promelas)
- Strain: n.a.
- Source: Commercial hatchery Aquatic Research Organisms, One Lafayette Road, Hampton, NH 03842, USA.
The fish were received at IMW-TNO on July 28, 1993 and kept in diluted DSWL at 22 °C until the start of the test.
- Age at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): The average standard length and weight of twenty fish of the batch used (± standard deviation) were 3.1 ± 0.1 cm and 0.46 ± 0.08 g respectively. The fish were randomly added to the test vessels.
- Weight at study initiation (mean and range, SD): see above (46 ± 0.08 g)
- Feeding: Not fed during the test.
- Other: The control media and test solutions were slightly aerated and replaced daily.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
4 d
Hardness:
< 180 mg/L as CaCO3
Test temperature:
21.0–21.4 °C
pH:
7.7–8.2
Dissolved oxygen:
≥ 6.7 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
0, 3.2, 5.6, 10, 18, 32, 56 mg/L (nominal)
0, 1.6, 3.4, 6.8, 13, 25, 56 mg/L (mean measured concentrations)
Details on test conditions:
TEST SYSTEM
- Test vessel: 12 L aquaria filled with 10 L medium
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: All-glass aquaria, each containing 10 litre of control or test medium.
- Aeration: Yes
- Type of flow-through (e.g. peristaltic or proportional diluter): n.a.
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates):2
- No. of vessels per vehicle control (replicates): n.a.
- Biomass loading rate: 0.46 ± 0.08 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used was diluted DSWL (Dutch Standard Water Linschoten), prepared from ground water.
- nominal composition:
Na+ = 1.19 mmol/L
K+ = 0.20 mmol/L
Ca2+ = 1.36 mmol/L
Mg2+ = 0.73 mmol/L
Cl- 2.72 = mmol/L
S042- 0.73 = mmol/L
HCO3- 1.39 = mmol/L
This medium is prepared by addition of several salts to groundwater from a locality near Linschoten (the Netherlands). The groundwater contains several other trace elements (< 1 mg/L). The equilibrium pH of the medium, after aeration, should be 8.3-8.5, but usually is slightly less, namely 8.0-8.2.
The total organic carbon content was 1.9 mg/L. For the present study DSWL was diluted with distilled water (volume ratio: DSW: distilled water = 4:1) to reach a hardness of < 180 mg/L.
- Intervals of water quality measurement: At the beginning and at the end of the test, as well as at each replacement time (in the spent solutions just before replacement and in the newly prepared solutions just after dosing), the pH, the oxygen concentration and the temperature of the control medium and the test solutions were measured. pH and oxygen concentrations were also measured after 3 h in the test solutions of 56 mg/L in which all fish were dead.
- All test solutions were completely clear (visually assessed) throughout the final test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : after 3 h, 6 h, 24 h, 30 h, 48 h, 54 h, 72 h, 78 h and 96 h the condition (swimming behaviour, colour, respiratory function and any other observable morphological or behavioural criterion) of the survivors was visually compared with that of the control animals.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.75
- Justification for using less concentrations than requested by guideline: n.a.
- Range finding study: yes, two range finding studies were performed
- Concentrations for the final test were determined in range-finding tests. The first range-finding test was carried out with the zebra fish Brachydanio rerio. Nominal concentrations of 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32, 100 and 320 mg/L were tested by dissolving 32, 100 and 320 mg of the test substance in one litre of diluted DSWL and subsequently dilution of these solutions. One test beaker containing about one litre of medium and five fish each were used for the control and each concentration. In the second range-finding test nominal concentrations of 3.2, 10 and 32 mg/L were tested with the fathead minnow Pimephales promelas by dissolving 100 mg of test substance in one litre of diluted DSWL and subsequently dilution of this solution. One test beaker containing about 1.5 litres and five fish were used for the control and each concentration.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
ca. 25 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: At 18 and 32 mg/L (nominal) fish with dark colour, disturbed equilibrium and slow swimming were observed after 6 h
- Observations on body length and weight: Not reported
- Other biological observations: Not reported
- Mortality of control: No mortality
- Other adverse effects control: No
- Abnormal responses: See above
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
n.a.
Reported statistics and error estimates:
See Table 2 below

Range-finding tests

In both range-finding tests all fish in the test media of 32 mg/L and higher were dead after one day of exposure. In the control and in all other test solutions the fish were alive. The condition of the fish in all other test media was equal to that of the controls.

After preparing an oily like layer was observed at the surface of the test medium of 320 mg/L. After one day exposure some turbidity was observed in the range-finding test with B. rerio at 10 mg/L and the higher concentrations tested. The pH values in the control and test media varied between 6.6 and 8.2 (range-finding test with B. rerio) and between 7.6 and 8.2 (range-finding test with P. promelas). The oxygen concentrations were 5.2 or higher (range-finding test with B. rerio) and 6.1 or higher (range-finding test with P. promelas).

 

Final test

pH

The pH varied between 7.7 and 8.2. It is not likely that the pH affected the results.

Oxygen concentration

The lowest value measured was 6.7 mg/L. It is not likely that the oxygen concentration affected the results.

Temperature

The temperatures of the control and test media varied between 21.0 and 21.4 °C. It is not likely that the temperature affected the results.

Observations on the test solutions

All test solutions were completely clear (visually assessed) throughout the test.

Chemical analysis

The results of the chemical analysis are given in Table 1.

Table 1: Results of chemical analysis of the test media.

Nominal

concentration of

test substance

Samples at t = 0 h

Samples at t = 3h

Samples at t = 24 h

Average

% of nominal

during

the test

(mg/L)

(mg/L)

% of nominal

(mg/L)

% of nominal

(mg/L)

% of nominal

 

0

<0.01

 

/

/

<0.01

 

 

3.2

2.33

73

/

/

0.92

29

51

5.6

4.35

78

/

/

2.27

41

60

10

9.1

91

/

/

4.38

44

68

18

17.2

96

/

/

8.7

48

72

32

31.5

98

/

/

19.0

59

78

56

58.2

104

54.5

97

/

29

100

 

Survival and other observations on the test fish

The results of the test, expressed as LC50, LC100 and NOEC values are given in Table 2. The observation on the mortality and condition of the fish are reported in Table 3.

There was no mortality in the control medium.

 

Table 2: Results of the test with 2-ethylhexyl lactate and Pimephales promelas.

Parameter

(h = hours)

Effect

Nominal concentration (mg/L)

(95% confidence interval)

Actual concentration (mg/L)

3h LC50

mortality

42 (32-56)

ca. 33

6h LC50

mortality

42 (32-56)

ca. 33

24h LC50

mortality

32 (32-46)

ca. 25

30h LC50

mortality

32 (32-46)

ca. 25

48h LC50

mortality

32 (32-46)

ca. 25

54h LC50

mortality

32 (32-46)

ca. 25

72h LC50

mortality

32 (32-44)

ca. 25

78h LC50

mortality

32 (32-44)

ca. 25

96h LC50

mortality

32 (32-44)

ca. 25

96hLC100

mortality

56

ca. 56

96h NOEC

mortality (= 96h LC0)

32 (Two fish were dead at 32 mg/L, but because 10% mortality (in the controls) is allowed, this concentration

is recorded as the 96h NOEC with regard to mortality as criterion.)

ca. 25

96h NOEC

Condition (slow swimming fish with disturbed equilibrium and dark color)

10

ca. 6.8 5'

Table 3: Number of surviving fish and their condition in the control and test media of 2-ethylhexyl lactate during the test (a and b are replicates).

Time [h]

Nominal concentration of the test substance [mg/L]

 

0

3.2

5.6

10

 

a

b

a

b

a

b

a

b

0

10

10

10

10

10

10

10

10

3

10

10

10

10

10

10

10

10

6

10

10

10

10

10

10

10

10

24

10

10

10

10

10

10

10

10

30

10

10

10

10

10

10

10

10

48

10

10

10

10

10

10

10

10

54

10

10

10

10

10

10

10

10

72

10

10

10

10

10

10

10

10

78

10

10

10

10

10

10

10

10

96

10

10

10

10

10

10

10

10

 

Time [h]

Nominal concentration of the test substance [mg/L]

 

18

32

56

 

a

b

a

b

a

b

0

10

10

10

10

10

10

3

10 a, c

10 a,c

10 a, b, c

10 a, b, c

0

0

6

10 a, b

10 a,b

10 a, b, c

9 a, b, c

0

0

24

10 a, b

10 a,b

10 a, b, c

9 a, b, c

0

0

30

10 b

10 b

10 a, b, c

9 a, b, c

0

0

48

10 b

10 b

10 a, b, c

9 a, b, c

0

0

54

10 b

10 b

10 a, b, c

9 a, b, c

0

0

72

10 b

10 b

10 a, b, c

9 a, b, c

0

0

78

10

10

10 a, b, c

9 a, b, c

0

0

96

10

10

10 a, b, c

9 a, b, c

0

0

 a) Slow swimming fish.

b) Disturbed equilibrium.

c) Fish with a dark colour observed

Validity criteria fulfilled:
yes
Remarks:
Zero control mortality, semi-static design hence sufficiently constant conditions, dissolved oxygen > 60 % saturation, results based on mean measured concentrations
Conclusions:
96-h LC50 = 25 mg/L actual concentration (32 mg/L nominal)
Executive summary:

In a 96-h acute toxicity study, fathead minnow (Pimephales promelas) were exposed to 2 -ethylhexyl-lactate at concentrations of 3.2, 5.6, 10, 18, 32, and 56 mg/L (nominal), corresponding to 1.6, 3.4, 6.8, 13, 25 and 56 mg/L (mean measured) under semi-static conditions (daily renewal).  The 96-h LC50 was 25 mg/L (measured), based on mortality.  Sublethal effects of slow swimming, disturbed equilibrium and dark colour were observed in the groups exposed to 13 and 25 mg/L (measured) of 2-ethylhexyl-lactate.  Based on the results of this study, 2-ethylhexyl-lactate would not be classified as toxic to fish (Pimephales promelas) in accordance with the classification system of the CLP Regulation.

This toxicity study is classified as acceptable and satisfies the guideline requirement for a short-term toxicity study in fish.

Results synopsis

Test organism size: 3.1 ± 0.1 cm

Test type: Static renewal

LC50:  25 mg/L

Endpoint(s) effected: Survival/mortality

Description of key information

The 96-h LC50 of 2-ethylhexyl-S-lactate in fish (Pimephales promelas) was determined to be 25 mg/L (measured).

Key value for chemical safety assessment

LC50 for freshwater fish:
25 mg/L

Additional information