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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:
26.10.1993-13.11.1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study has been performed according to OECD guideline. Reliability is restricted by missing information on stability of test substance. However, based on availble stability data of 1H-Benzotriazole test concentration > 80 % during the whole test period is expected.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Strain:
- Source: M.B. Ruysbroeck B.V. (Noordvliet 159, Maassluis)
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): 2.7 ± 0.2 cm
- Weight at study initiation (mean and range, SD): 0.11 ± 0.02 g
- Method of breeding:
- Feeding during test
- Food type: fishes were not fed
- Amount: -
- Frequency: -

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
- Duration:
- Health/mortality:
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
210 mg/L (based on CaCO3)
Test temperature:
24.3 - 25.4 °C
pH:
7.2 - 8.2
Dissolved oxygen:
> 6.6 mg/L
Nominal and measured concentrations:
32, 56, 100, 180, and 320 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 2 L beaker
- Material, size, headspace, fill volume: all-glass, 1.5 L medium
- Aeration: slightly aerated
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 10 fishes
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment
- Photoperiod: 16 / 8 (light /dark)
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality, behaviour (0, 24, 48, 72, 96 h)

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
240 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 180-320 mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
240 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 180-320 mg/L
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
180 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 163-221 mg/L
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
180 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 154-210 mg/L
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
320 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality

 

Time (h)

Nominal concentration of test substance (mg/L)

0

32

56

100

180

320

0

10 (a)

10 (a)

10 (a)

10 (a)

10 (a)

10 (a)

24

10 (a)

10 (b)

10 (b)

10 (b)

10 (c)

10 (d)

48

10 (a)

10 (b)

10 (b)

10 (b)

10 (c)

0

72

10 (a)

10 (b)

10 (b)

10 (b)

6 (c)

0

 

 

 

 

 

4 (d)

 

96

10 (a)

10 (b)

10 (b)

10 (b)

5 (c)

0

 

 

 

 

 

1 (d)

 

 

(a)  Condition of all fish, visually assessed, normal (=good).

(b)  Condition of all fish, visually assessed, equal to that of the control fish

(c)   Number of fish with disturbed equilibrium

(d)  Number of dead fish

Validity criteria fulfilled:
yes
Remarks:
Although test substance concentration was not analytically monitored during semi-static test stable concentrations (> 80 % nominal concentration) were assumed due to stability data of the substance
Conclusions:
Based on experimental observations an effect concentration LC50(96 h) = 180 mg/L (95 % c.i. 154 - 210 mg/L) has been calculated. Notwithstanding the uncertainties with regard to the substance stability, the study has been considered reliable and adequate for regulatory purposes.
Executive summary:

The acute toxicity of the test substance to the fresh-water fish species Brachydanio rerio was determined in an OECD guideline study according to GLP. The test was carried out under semi-static conditions with daily replacement of the test solutions and with 10 fish for the control medium and each concentration. The exposure duration was 96 hours.

The nominal concentrations tested were 32, 56, 100, 180, and 320 mg/L. All test solutions were completely clear (visually assessed) throughout the test.

Endpoint:
short-term toxicity to fish
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Experimental Start Date: 16/6/03; Experimental Completion Date: 21/6/03
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
1H-Benzotriazole is expected to show an effect concentration in a similar range like the analogue Tolyltriazole havs shown in several available studies due to a related mode of action and a similar physico-chemical profile. The source chemical is sufficient similar to read across towards 1H-Benzotriazole.
Qualifier:
according to
Guideline:
other: The test procedure is based on test guideline PARCOM 1995 Part B Protocol for a Fish Acute Toxicity Test (modified OECD 203 Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Samples were not taken for analytical measurement of test substance concentration in the test samples.
Vehicle:
no
Details on test solutions:
The test substance as thoroughly homogenised before use. The test substance was insoluble in water, and the sample preparation method for water insoluble test substances was followed. The water accommodated fraction (WAF) of the test substance was tested. Standard sample preparation methods are described below:

Mixtures of non-water soluble test substances contain components of varying water solubility. Hence, each test substance concentration is prepared separately to avoid problems with respect to solubility.
- For each test concentration mix the test substance with 5 litres of reference seawater for ~20 hours. Settle for 1-4 hours and add 3 litres, of the WAF, of each dose directly to the test vessels.
- Add 5 times the final test substance concentration.

This procedure is repeated after 48 hours during the 96 hour test.

Based on the results from the Skeletonema costatum preliminary test, the following test concentrations (nominal concentrations of whole test substance) were used for the definitive test: 10, 30, 100, 300 and 1000 mg/l.
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISM
- Source: Sea Platation Engineering Inc.
- Age at study initiation (mean and range, SD): Juvenile
- Length at study initiation (length definition, mean, range and SD): 1.6 - 2.0 cm, average 1.8 cm
- Weight at study initiation (mean and range, SD): 0.0440 - 0.1324 g, average 0.0835 g


ACCLIMATION
Holding conditions: Synthetic/Natural seawater; 17.6 - 22.4ºC, salinity: 33%, dissolved oxygen >60%; semi-static
On arrival at the laboratory, the fish were transferred to a temperature room to allow temperature acclimatisation. Temperature in the fish containers and the acclimatisation tank were measured. The fish were held in a seawater flow-through system, and pH, salinity, O2 saturation and temperature were measured within one hour of arrival and subsequently on every working day. Aeration was supplied and the fish were fed at least once per working day. Mortality during the first 9 days after arrival was 0%. This was within the valid range.
Test type:
semi-static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable.
Hardness:
Not stated.
Test temperature:
Temperature in test solutions ranged from 19.2 - 20.1ºC
pH:
pH in test solutions ranged from 7.47 - 8.21
Dissolved oxygen:
Dissolved oxygen in test solutions was ≥98%
Salinity:
Salinity in test solutions ranged from 32.2 - 33.2%
Nominal and measured concentrations:
Definitive test: Nominal concentrations (nominal whole substance): 10, 30, 100, 300 and 1000 mg/l.
Details on test conditions:
TEST SYSTEM
- Renewal rate of test solution (frequency/flow rate): Test media changed after 48 hours.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per vehicle control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
Reference seawater used for testing was collected from Bjolsen Akvarium AS and was stored for no longer than 4 weeks at temperatures 20.0 ± 2.4ºC

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The fish were inspected at least once per day during the whole test period. Fish that were considered dead were removed from the test vessels and mortality was recorded. Behaviour was also observed during the test period.

Measurements of pH, dissolved oxygen, salinity and temperature were carried out daily in all test vessels including the control.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(whole test substance)
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(whole test substance)
Basis for effect:
mortality
Details on results:
Log test substance concentrations, mortality, LC50 values and variables are shown in Tables 1 and 2.

The LC50 value for the test substance R-2191 was found to be 55 mg/L after 96 hours.
The 95% confidence range could not be calculated.

The NOEC was estimated to be 30 mg/L.

LC50 values after 24, 48 and 72 hours were 174, 118 and 55 mg/L respectively.


Results with reference substance (positive control):
A reference test using a single concentration of 2.3 mg/L of 3,5-dichlorophenol was performed. The recorded mortality was 40% after 96 hours.

Table 1. Log Test Substance Concentrations and Mortality after 24, 48, 72 and 96 Hours

Test Conc (mg/L)

Log Test Conc

No. fish per vessel

Mortality 24 h

Mortality 48 h

Mortality 72 h

Mortality 96 h

Cumulative mortality %

Control

-

10

0

0

0

0

0

10

1.0

10

0

0

0

0

0

30

1.48

10

0

0

0

0

0

100

2.00

10

0

1

10

10

100

300

2.48

10

10

10

10

10

100

1000

3.00

10

10

10

10

10

100

Table 2. LC50 values and thr 95% Confidence Limits after 24, 48, 72 and 96 hours for the Test Substance R-2191

Duration (hours)

LC50 (mg/L)

Lower 95% CL (mg/L)

Upper 95% CL (mg/L)

24

174

-

-

48

118

-

-

72

55

-

-

96

55

-

-

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 value for the test substance R-2191 was found to be 55 mg/L (nominal whole test substance). The NOEC value was estimated as 30 mg/L (nominal whole test substance). Study has been considered adequate for regulatory purposes.
Executive summary:

Acute toxicity (96 h LC50: semi-static test) of the test substance R-2191 to the juvenile Sheepshead minnow, Cyprinodon variegatus, has been assessed in accordance with test guideline PARCOM 1995 Part B (modified OECD Guideline 203 "Fish, acute toxicity test").

The tested concentrations were 10, 30, 100, 300 and 1000 mg/L (nominal whole test substance).

The 96 hour LC50 value for the test substance R-2191 was found to be 55 mg/L (nominal whole test substance). The 95% confidence limit could not be calculated. The NOEC value was estimated to be 30 mg/L (nominal whole test substance).

In the test the control parameters for temperature, dissolved oxygen and salinity were within the recommended ranges.

Description of key information

Key value for freshwater was selected from OECD guideline 203 study with Danio rerio. Key value for marine environment was selected from modified 203 study with Cyprinodon variegatus.

Key value for chemical safety assessment

LC50 for freshwater fish:
180 mg/L
LC50 for marine water fish:
55 mg/L

Additional information

Two short-term freshwater studies with Brachydanio rerio were available showing an effect value LC50 more than 100 mg/L. Based on these results and the results from the test in the other trophic levels fish as higher tier consument is considered as least sensitive to benzotriazoles. Available marine water study from read-across with analogue Tolyltriazole indicate moderate toxicity towards fishes, too.