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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:
1996-11-20 - 2020-12-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
„Acute Toxicity for Fish" Council Directive 92/69/EEC C.l (1992)
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
not specified
Principles of method if other than guideline:
Only three different concentrations have been tested instead of at least five concentrations differing by a constant factor not exceeding 2.2, as given in OECD Guideline 203 and in EU Method C.1.
GLP compliance:
not specified
Analytical monitoring:
no
Details on sampling:
Not applicable.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was added to 5 liters of dilution water (direct weighing) and treated for 60 seconds with an ultra turrax and afterwards stirred for 1 hour on a magnetic stirrer. Finally undissolved particles of the test substance were removed by filtration with a folded filter.
- Controls: Synthetic fresh water according to ISO 7346
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish (Danio rerio)
- Length at study initiation (length definition, mean, range and SD): 2.5 - 3.5 cm
- Medical pre-treatment: none
- Method of breeding: not specified

ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions (same as test or not): The animals are kept in synthetic fresh water according to ISO 7346.
- Type and amount of food during acclimation: not specified
- Feeding frequency during acclimation: not specified
- Health during acclimation (any mortality observed): < 2% mortality while kept
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No information available.
Hardness:
No information available.
Test temperature:
Control: 21.6 - 21.9 °C
1000 mg/L: 21.8 - 22.0 °C
3160 mg/L: 21.2 - 21.9 °C
10000 mg/L: 21.4 - 21.7 °C
pH:
Control: 7.4 - 7.9
1000 mg/L: 7.5 - 7.8
3160 mg/L: 7.2 - 7.7
10000 mg/L: 7.3 - 7.7
Dissolved oxygen:
Control: 8.0 - 8.8 mg/L (93.5 - 102.4 % ASV)
1000 mg/L: 8.0 - 8.9 mg/L (93.8 - 104.3 % ASV)
3160 mg/L: 8.4 - 8.9 mg/L (98.2 - 103.4 % ASV)
10000 mg/L: 8.7 - 8.8 mg/L (100.9 - 102.6 % ASV)
Salinity:
No information available.
Nominal and measured concentrations:
Nominal: 1000, 3160 and 10000 mg/L and a control
Details on test conditions:
TEST SYSTEM
- Test vessel: vessels of 300 x 135 x 200 mm
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 5 L test medium
- Aeration: not specified
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic fresh water according to ISO 7346.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: not specified
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and visible effects on appearance and behaviour of the test fish were determined and recorded after 0, 2, 24, 48, 72 and 96 hours of test duration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
no
Remarks:
Not required under OECD 203, not least for animal welfare reasons.
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
3 160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
5 621 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: geometric mean of the nominal LC0 and LC100 (calculated)
Results with reference substance (positive control):
Not relevant.
Reported statistics and error estimates:
No statistical analysis was performed. The LC0 and the LC100 are determined directly from the raw data and the LC50 is calculated as geometric mean of these two concentrations or read from the dose-response curve.

Study Evaluation

Mortality

Concentration of the test substance: Control

2 h

24 h

48 h

72 h

96 h

Absolute

0

0

0

0

0

Cummulative

0

0

0

0

0

Cummulative [%]

0

0

0

0

0

Concentration of the test substance: 1000 mg/L

2 h

24 h

48 h

72 h

96 h

Absolute

0

0

0

0

0

Cummulative

0

0

0

0

0

Cummulative [%]

0

0

0

0

0

Concentration of the test substance: 3160 mg/L

2 h

24 h

48 h

72 h

96 h

Absolute

0

0

0

0

0

Cummulative

0

0

0

0

0

Cummulative [%]

0

0

0

0

0

Concentration of the test substance: 10000 mg/L

2 h

24 h

48 h

72 h

96 h

Absolute

0

0

-

-

-

Cummulative

0

10

-

-

-

Cummulative [%]

0

100

-

-

-

A defined concentration of the test substance will lead to a certain percentage proportion of dead fish at the end of the study period.

The LC0 and the LC100 are determined directly from the study, while the LC50 is calculated or read from the dose-response curve. The study lasts for 96 hours.

Visible effects on appearance and behaviour of fish

Concentration of the test substance : Control

Symptoms of the test fish (2 h - 96 h): 10 animals normal swimming action*

Concentration of the test substance : 1000 mg/L

Symptoms of the test fish (2 h - 96 h): 10 animals normal swimming action

Concentration of the test substance : 3160 mg/L

Symptoms of the test fish (2 h - 96 h): 10 animals normal swimming action

Concentration of the test substance : 10000 mg/L

Symptoms of the test fish (2 h): 10 animals normal swimming action

*Catalogue:

1 normal swimming action

2 darting, uncontrolled swimming action

3 hectic, excited swimming action

4 gasping for air; irregular breathing

5 accelerated breathing

6 abnormal swimming action

7 sluggish swimming action

8 increased aggression

9 leaping action

10 uncontrolled collision with aquarium walls

11 motionless; uncontrolled swimming action when touched

12 symptoms could not be determined due to the strong coloration of test media

Validity criteria fulfilled:
yes
Conclusions:
The study was performed according to EU Method C.1 (which is in most parts equivalent to OECD guideline 203) without deviations which may have an impact on the validity of the study. Thus, the results were obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable. Under the conditions used for the test, the 96h LC0 was determined to be 3160 mg/L, the 96h LC50 was determined to be 5621 mg/L and the 96h LC100 was determined to be 10000 mg/L.
Based on these results, the test item does not need to be classified as toxic to freshwater fish following the provisions laid down in Council Directive 67/548/EEC and Regulation (EC) No. 1272/2008.
Executive summary:

The acute toxicity of the test substance to fish was investigeted according to EU Method C.1 (which is in most parts equivalent to OECD guideline 203). No GLP compliance was cited but the test facility is known to work under standard good laboratory practice. Danio rerio (Zebra fish) was used as test organism and exposed to nominal test concentrations of 1000, 3160 and 10000 mg/L of the test substance for 96 hours under static conditions. A control was running in parallel containing dilution water only. The test was carried out using a static design with a temperature range of 21.2 °C - 22.0 °C (control: 21.6 °C - 21.9 °C) and a pH range of 7.2 - 7.8 (control: 7.4 - 7.9) depending on the substance concentrations (1000, 3160 and 10000 mg/L). The light-dark cycle was 16 hours light / 8 h dark. Direct weighing was the method of administration followed by stirring and filtration. Any mortality and / or visisble sublethal effects were observed and recorded throughout the test.

The 96h LC0 and the 96h LC100 were determined directly from the raw data and reported to be 3160 and 10000 mg/L, respectively. The 96h LC50 was calculated as geometric mean of the 96h LC0 and the 96h LC100 and was reported to be 5621 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented publication, which meets basic scientific principles
Qualifier:
according to
Guideline:
other: Committee on Methods for Toxicity Tests with Aquatic Organisms (1975) Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians. U.S. EPA. Ecological Research Series EPA-660/3-75-009.
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Water samples (100 mL) taken at the beginning and end of each test were analysed for toxicant concentration. Precipitation, volatility, photolysis, adsorption to surfaces, and reactivity with water are factors that possibly affect the toxicant concentration in non-renewed static bioassays. Documentation of these effects is important to an understanding of the toxic processes of each chemical.
Vehicle:
yes
Details on test solutions:
Toxicants were administered either directly or in the form of stock solutions in deionized water or acetone. The solutions were briefly stirred with a glass rod, then a water sample was withdrawn from one tank at each concentration for quantitative toxicant analysis.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnows (Pimephales promelas)
- Source: EPA Newtown Fish Toxicology Station, Cincinnati, Ohio
- Length at study initiation (length definition, mean, range and SD): 3.2 to 4.2 cm standard length
- Feeding during test: no, only at maintenance before
- Food type: Tetra SM (Tetra-Werke, W. Germany)
- Amount: a twice daily ration
- Frequency: twice daily

ACCLIMATION
- Acclimation period: The minnows were acclimated to the test dilution water quality and temperature over the first two days of a four-day acclimation period. Solution withdrawal and renewal twice daily accomplished the water quality transition and temperature was reduced from 25 to 22 °C with the aid of a temperature bath-circulator. The fish were held for the last two days of acclimation at 22 °C in 100 % dilution water, and no food was given during the four-day period.

Other:
- An initial prophylactic treatment for external parasites consisting of 5 mg/L potassium permanganate was administered on three consecutive days, then hygienic conditions were maintained by sterilizing all fish care equipment with a 200 mg/L calcium hypochlorite (HTH) solution. The fish were observed for a minimum of 14 days (at least 10 days after any disease treatment) before being acclimated for testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No details available.
Hardness:
hardness: 40-48mg/L as CaC03;
alkalinity: 30-35 mg/L as CaC03;
conductivity: 120-160 µohm/cm
Test temperature:
22 C° +/- 1 °C
pH:
7.2 - 7.9
Dissolved oxygen:
No details available.
Salinity:
No details available.
Nominal and measured concentrations:
Not reported in detail. The concentrations tested were arranged in geometric series with at least a 60 % dilution factor.
Details on test conditions:
TEST SYSTEM
- Test vessel: circular fibre glass tanks
- Type: open
- Material, size, headspace, fill volume: 12 L, depth of aproximately 24 cm
- Aeration: the water was intensely aerated prior to use, but not during the test duration.
- No. of organisms per vessel: Five organisms were placed in each of two duplicate aquaria.
- No. of vessels per concentration (replicates): a total of 10 organisms tested per concentration.
- No. of vessels per control (replicates): At least two control aquaria were run. Control and solvent control mortality were less than 10% in all cases.
- No. of vessels per vehicle control (replicates): with additional control aquaria for any solvents used to introduce the toxicant into the test solutions. Control and solvent control mortality were less than 10 % in all cases.
- Biomass loading rate: less than 0.8 g/L

OTHER TEST CONDITIONS
- Photoperiod: 16 h light-8 h dark diurnal cycle
Reference substance (positive control):
not required
Remarks:
no specific reference substance was used, different chemicals were tested.
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
194.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 143.8 - 278.6
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
172.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 118.1 - 250.4
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
164.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 108.8 - 240.4
Details on results:
No further details are available.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
Probit analysis (Finney, 1971; Barr et al., 1976)
Moving average method
Bionominal test (Stephan, 1977)
Linear regression analysis
Calculation of an arbitrary limit
Table 2. LC50s and 95 % confidence intervals (mg/L)based on nominal concentrations for bioassays with the freshwater fathead minnow Pimephales promelas
  24h 48h 96h
Compound LC50 95%C.l. LC50 95%C.I. LC50 95%C.l.
Ammonium fluoride 438.5 348.7-575.8 417.5 331.3-542.5 363.8 295.7-507.3
Arsenic trisulfide * 644.3 493.1-1010.9 135.1 107.3-173.6
Benzoyl chloride 42.6 35.4-57.4 34.7 28.5-45.3 34.7 28.5-45.3
Benzyl chloride 11.6 8.9-14.6 7.3 5.1-10.5 (4.7-7.8)+
Cupric acetate 0.48 0.37-0.66 0.42 0.33-0.56 0.39 0.30-0.51
o-Dichlorobenzene (77.8-129.9)+ 76.3 60.7-102.6 57.0 42.6-80.4
p-Dichlorobenzene 35.4 30.5-43.0 35.4 30.5-43.0 33.7 29.0-40.4
Mercuric acetate 0.53 0.43-0.69 0.42 0.34-0.56 0.19 0.04-0.30
Mercuric thiocyanate 0.39 0.31-0.51 0.39 0.31-0.51 0.15 0.08-0.22
Resorcinol 88.6 72.3-113.6 72.6 58.7-92.3 53.4 41.1-72.1
Sodium hypochlorite # 5.6 4.4-7.1 5.9 4.8-8.0 5.9 4.8-8.0
Toluene-2,4-diisocyanate 194.9 143.8-278.6 172.1 118.1-250.4 164.5 108.8-240.4
* No significant mortality (<65 %).
 + Based on one partial kill, the best information that the binomial test gives is that we are 82.7 %, confident that LC50 lies in this interval.
# 4-6 % aqueous solution (Fisher Scientific).

All values shown in these tables were computed on the basis of the nominal concentrations. The physicochemical behavior of the compounds had a major impact upon their toxicity to aquatic organisms.

Toluene-2,4-di-isocyanate (TDI) reacted with the dilution water to form carbon dioxide and a polyurethane foam-like solid (Roberts & Caserio, 1965). The TDI appeared to be toxic to fathead minnows only in unreacted form, since all mortalities occurred during the first 12 h of the test. A concurrent decrease in pH was observed as a result of carbon dioxide formation.

Analytical measurements of toxicant were made primarily to document the visual observations of chemical behavior.

Good correspondence between measured and nominal concentrations was entirely dependent on total solubility of the toxicant, and its homogeneous dispersal in the aquaria. There is essentially no value in using measured concentrations to compute LC50's unless one can be sure that the measured concentration represents the entire exposure situation.

For pragmatic reasons, the nominal concentrations tested are essential information to future researchers; duplication of measured concentration would be difficult at best without knowledge of the initial toxicant input. This is not to denigrate the value of analytical measurement of toxicant, since this research clearly showed the importance of documenting physicochemical behavior.

Validity criteria fulfilled:
yes
Remarks:
Basic scientific principles were met.
Conclusions:
This report is regarded as a valid paper, which meets basic scientific principles to test the acute toxicity of a substance to fish. According to the reported 96h LC50 value (> 100 mg/L), the test substance has not to be classified as toxic to freshwater fish.
Executive summary:

The study is supported by an acute toxicity study on fish conducted with the read-across substance Toluene diisocyanate (TDI, CAS 26471 -62 -5). In this study, Pimephales promelas was chosen as freshwater representative and was tested under static conditions according to the Commitee on Methods for Toxicity Tests with Aquatic Organisms (1975) Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians. U.S. EPA. Ecological Research Series EPA-660/3-75-009 (Curtis et al., 1978). The basic scientific principles of fish tests as performed today are met. The test duration was 96 hours at a temperature of 22 +/- 1 °C without aeration or feeding during the test period. A series of substances were tested, whereby the substances were administered either directly or in the form of stock solutions in deionized water or acetone. The solutions were briefly stirred with a glass rod, then a water sample was withdrawn from one tank at each concentration for quantitative toxicant analysis. The test concentrations were arranged in geometric series with at least a 60 % dilution factor. As result, a 96h LC50 value of 164.5 mg/L with 95 % confidence limits of 108.8 - 240.4 mg/L is reported.

Description of key information

1) Key_Short-term toxicity to fish: LC50(96h) = 5621 mg/L (nominal) for Danio rerio (static, freshwater, EU Method C.1)
2) Supporting_Short-term toxicity to fish_RA TDI: LC50 (96h) = 164.5 mg/L for Pimephalis Promelas (static, freshwater, EPA-660/3-75-009)

Key value for chemical safety assessment

LC50 for freshwater fish:
5 621 mg/L

Additional information

The acute toxicity of the test substance to fish was investigeted according to EU Method C.1 (which is in most parts equivalent to OECD guideline 203). No GLP compliance was cited but the test facility is known to work under standard good laboratory practice. Danio rerio (Zebra fish) was used as test organism and exposed to nominal test concentrations of 1000, 3160 and 10000 mg/L of the test substance for 96 hours under static conditions. A control was running in parallel containing dilution water only. The test was carried out using a static design with a temperature range of 21.2 °C - 22.0 °C (control: 21.6 °C - 21.9 °C) and a pH range of 7.2 - 7.8 (control: 7.4 - 7.9) depending on the substance concentrations (1000, 3160 and 10000 mg/L). The light-dark cycle was 16 hours light / 8 h dark. Direct weighing was the method of administration followed by stirring and filtration. Any mortality and / or visisble sublethal effects were observed and recorded throughout the test.

The 96h LC0 and the 96h LC100 were determined directly from the raw data and reported to be 3160 and 10000 mg/L, respectively. The 96h LC50 was calculated as geometric mean of the 96h LC0 and the 96h LC100 and was reported to be 5621 mg/L.

The study is supported by an acute toxicity study on fish conducted with the read-across substance Toluene diisocyanate (TDI, CAS 26471 -62 -5). In this study,Pimephales promelas (Fathead minnow) was chosen as freshwater representative and was tested under static conditions according to the Commitee on Methods for Toxicity Tests with Aquatic Organisms (1975) Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians. U.S. EPA. Ecological Research Series EPA-660/3-75-009 (Curtis et al., 1978). The basic scientific principles of fish tests as performed today are met. The test duration was 96 hours at a temperature of 22 +/- 1 °C without aeration or feeding during the test period. A series of substances were tested, whereby the substances were administered either directly or in the form of stock solutions in deionized water or acetone. The solutions were briefly stirred with a glass rod, then a water sample was withdrawn from one tank at each concentration for quantitative toxicant analysis. The test concentrations were arranged in geometric series with at least a 60 % dilution factor. As result, a 96h LC50 value of 164.5 mg/L with 95 % confidence limits of 108.8 - 240.4 mg/L is reported.

. .