Registration Dossier

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-11-17 to 1997-11-21 (range-finding) and 1998-05-11 to 1998-05-15 (study)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: each stock solution and test sample
- Sampling method: quantitative analysis of water samples (fresh test media) at 0, 24, 48, 72, 96 hours
- Sample storage conditions before analysis: immediate analysis after preparation of the test samples
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Controls: control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Ultrasonification for between 30 and 60 seconds;
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): final vehicle concentration in test solutions =1 mL/L; solvent control = 100 µg/L of dimethylformamide
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no (range-finding study showed precipipation of TODI in access of 4.0 mg/L which was the highest test concentration)
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Parkwood Trout Farm, Harrietsham, Kent, UK
- Age at study initiation: juvenile
- Length at study initiation: 4.8 cm (sd=0.4)

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): no mortality observed

FEEDING:
- Feeding during test: until 24 hours prior to the start of the definitive study
- Food type: commercial trout pellets
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
96 h
Hardness:
100 mg/L CaCO3
Test temperature:
14°C ± 1°C
pH:
pH 7.2 (24 h) to 7.7 (96 h)
Dissolved oxygen:
Greater than or equal 9.4 mg O2/L
Salinity:
Conductivity: 468 µS/cm
Nominal and measured concentrations:
- nominal concentration: 0.40, 0.72, 1.3, 2.2, 4.0 mg/L
- time-weight mean measured concentration: 0.0079, 0.178, 0.294, 0.566, 0.662 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: closed (by cover)
- Material, size, headspace, fill volume: 20 litre glass vessels were filled with 20 litre water
- Aeration: aearated via narrow bore glass tubes
- Type of flow-through: none, semi-static (see below)
- Renewal rate of test solution (frequency/flow rate): daily renewal of the test preparations to maintain concentrations of the test material as near to nominal as possible (due to instantaneouse hydrolysis)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): one vessel per concentration
- No. of vessels per control (replicates): one
- No. of vessels per vehicle control (replicates): one
- Biomass loading rate: 0.82 g body weight/litre


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water (dechlorinated and partly softenend)
- Total organic carbon: 1.2 mg/L
- Particulate matter: < 0.5 Formazin Turbidity Unit (F.T.U.)
- Pesticides: < 0.03 µg/L
- Toatal Chlorine: 0.11 mg/L
- Alkalinity: 137 mg/L
- Conductivity:468 µS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED:
- Mortalities and sub-lethal effects were recorded at 3, 6, 24, 48, 72 and 96 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 1.0 and 4.0 mg/L
- Results used to determine the conditions for the definitive study: There were no sub-lethal effects of exposure during the range-finding study. The results showed no mortalities at the test concentration of 1.0 mg/L. However, mortalities were observed at 4.0 mg/L. During preliminary solubility work precipitation of test material was observed (by visual inspection) at concentrations in excess of 4.0 mg/L indicating this to be the maximum attainable test concentration under test conditions.
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.25 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities:
at 1.3 mg/L (nominal conc.): swimming at bottom after 6 hour and onwards;
at 2.2 mg/L swimming at bottom and loss of equilibrium at 3 to 24 hours; at 48 hours all fish dead;
at 4.0 mg/L swimming at bottom and loss of equilibrium at 3 to 6 hours; at 24 hours all fish dead
- Observations on body length and weight: None: mean standard length of 4.8 cm (sd=4) and a mean weight of 1.64 g (sd=0.44)
- Mortality of control: none
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on nominal test concentrations gave as 96-hour LC50 value of 1.0 mg/L with 95 % confidence limits of 0.90 - 1.1 mg/L.
The No Observed Effect Concentration was 0.72 mg/L.
The 96 -hour LC50 value based on time-weighted-mean measured concentrations was 0.25 mg/L with 95% confidence limits of 0.22 - 0.28 mg/L.
The No Observed Effect Concentration was 0.18 mg/L.
Despite the deviations from the nominal concentrations it was considered justifiable to base the results on time-weighted-mean measured concentrations also.
Nevertheless, this acute toxicity test in fish represents a worst case as the solubility of TODI (and its degradation / hydrolysis product) in water was increased by an auxiliary solvent and the study result is based on time-weighted-mean measured concentrations which can also be regarded as a further overestimation as LC50 refers to a substance concentration which was reduced due to hydrolysis. Actually, it can be assumed that remaining substance and (!) degradation product are responsible for the effects. Thus, potentially in total 1 mg consisting of TODI and its hydrolysis product per litre are responsible for the effects. Studies on LC50 in fish only considering the toxicological potential of TODI but not of its product of hydrolysis are not possible.
Executive summary:

Methods

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedures

Following a preliminary range-finding study, fish were exposed, in groups of 10, to an aqueous dispersion of the test material over a range of concentrations of 0.40, 0.72, 1.3, 2.2 and 4.0 mg/L for a period of 96 hours under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the study until termination after 96 hours.

Results

The 96 -Hour LC50 based on nominal test concentrations was 1.0 mg/L with 95% confidence limits of 0.90 - 1.1 mg/L. The No Observed Effect Concentration was 0.72 mg/L.

The test concentration of 4.0 mg/L was the highest attainable test concentration due to the limited solubility of the test material in water and auxiliary solvent, and having due regard for the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Pre-study stability analysis performed, showed the test material to be highly unstable in aqueous media, with hydrolysis occurring virtually instantaneously on contact with water. The degradation product was found to be insoluble in organic and aqueous solvents, thus negating the possibility of quantitative analysis of the degradant.

Therefore, the freshly prepared stock solutions and test samples were extracted and analysed immediately after preparation at 0, 24, 48 and 72 hours. Given the extremely rapid hydrolysis of the test material, samples of the expired test media were analysed at 24 and 96 hours only.

Analysis of the solvent stock solutions showed measured concentrations ranging from 57% to 106% of nominal, the results from analysis of the freshly prepared test samples showed measured values ranging from 8% to 91% of nominal. Analysis of the expired test media gave measured values which ranged from less than the limit of quantitation to 24% of nominal.

The results show, that despite immediate extraction and analysis of the freshly prepared stock solutions and test samples, variation in the measured concentrations was unavoidable due to the rapid hydrolysis of the test material. Further hydrolysis was shown to occur over each 24 hour test media renewal period and so it was considered justifiable to base the results on time-weighted-mean measured concentrations also.

The 96 -hour LC50 value based on time-weighted-mean measured concentrations was 0.25 mg/L with 95% confidence limits of 0.22 - 0.28 mg/L. The No Observed Effect Concentration was 0.18 mg/L.

Nevertheless, this acute toxicity test in fish represents a worst case as the solubility of TODI (and its degradation / hydrolysis product) in water was increased by an auxiliary solvent and the study result is based on time-weighted-mean measured concentrations which can also be regarded as a further overestimation as LC50 refers to a substance concentration which was reduced due to hydrolysis. Actually, it can be assumed that remaining substance and (!) degradation product are responsible for the effects. Thus, potentially in total 1 mg consisting of TODI and its hydrolysis product per litre are responsible for the effects. Studies on LC50 in fish only considering the toxicological potential of TODI but not of its product of hydrolysis are not possible.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
not specified
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
Each water sample of 100 mL was removed from each test vessel at 0 and 24 hours (before water change). Immediately the sample was analyzed by HPLC method.
Vehicle:
not specified
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Common name: Killifish

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24 +/- 1
Nominal and measured concentrations:
nominal concentrations: 7.5, 10, 13, 18, 24, 32, 42 and 56 mg/L
measured concentrations: 7.05, 9.58, 12.3, 16.7, 22.7, 30.0, 40.6, 53.7 mg/L
Details on test conditions:
TEST SYSTEM
- Fill volume: 4.0 L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light, 8-hour dark
- Light intensity: room light

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.3

Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Reported statistics and error estimates:
Probit method
Validity criteria fulfilled:
yes
Conclusions:
The 96 -hour LC50 based on nominal test concentrations was 13.0 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of TODI's degradation product (4,4'-bi-o-toluidine) to Killifish (Oryzias latipes) as requested by ECHA. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test".

Fish were exposed, in groups of 10 to the test material over a range of concentrations of 7.5, 10, 13, 18, 24, 32, 42 and 56 mg/L for a period of 96 hours under semi-static test conditions.

Water sample was taken from each test vessel at 0 hour and 24 hours and immediately analyzed by HPLC method. On average, the recovery rate was 98.3% at 5 mg/L and 97.7% at 60 mg/L.

 

The 96 -hour LC50 based on nominal test concentrations was 13.0 mg/L with 95% confidence limits of 11 - 16 mg/L.

 

Description of key information

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on nominal test concentrations gave as 96-hour LC50 value of 1.0 mg/L with 95 % confidence limits of 0.90 - 1.1 mg/L. The No Observed Effect Concentration (NOEC) was 0.72 mg/L.
The 96 -hour LC50 value based on time-weighted-mean measured concentrations was 0.25 mg/L with 95% confidence limits of 0.22 - 0.28 mg/L. The NOEC was determined to be 0.18 mg/L.

 

Key value for chemical safety assessment

LC50 for freshwater fish:
0.25 mg/L

Additional information

Key study

The acute toxicity of TODI to the freshwater fish rainbow trout (Oncorhynchus mykiss) was investigated according to an OECD 203 guideline.

The test item was presented in dimethylformamide to allow the formation of a stable dispersion. Following a preliminary range-finding study, juvenile fish were exposed, in groups of 10, to an aqueous dispersion of the test material over a range of concentrations of 0.40, 0.72, 1.3, 2.2 and 4.0 mg/L for a period of 96 hours under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the study until termination after 96 hours. In this 96-hour semi-static acute toxicity test with Rainbow trout the 96 h LC50 value was 1.0 mg/L (95% confidence limits: 0.90 - 1.1 mg/L), and the 96 h NOEC was determined to be 0.72 mg/L based on nominal concentrations.

The 96 -hour LC50 value based on time-weighted-mean measured concentrations was 0.25 mg/L with 95% confidence limits of 0.22 - 0.28 mg/L. The No Observed Effect Concentration was 0.18 mg/L.

Despite the deviations from the nominal concentrations it was considered justifiable to base the results on time-weighted-mean measured concentrations also.

Nevertheless, the acute toxicity test in fish represents a worst case as the solubility of TODI (and its degradation / hydrolysis product) in water was increased by an auxiliary solvent and the study result is based on time-weighted-mean measured concentrations which can also be regarded as a further overestimation as LC50 refers to a substance concentration which was reduced due to hydrolysis.

 

Supporting study

 A study was performed to assess the acute toxicity of the degradation product ( 4,4'-bi-o-toluidine) to Killifish (Oryzias latipes). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test".

Fish were exposed, in groups of 10 to the test material over a range of concentrations of 7.5, 10, 13, 18, 24, 32, 42 and 56 mg/L for a period of 96 hours under semi-static test conditions.

Water sample was taken from each test vessel at 0 hour and 24 hours and immediately analyzed by HPLC method. On average, the recovery rate was 98.3% at 5 mg/L and 97.7% at 60 mg/L.

 

The 96 -hour LC50 based on nominal test concentrations was 13.0 mg/L with 95% confidence limits of 11 - 16 mg/L.