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Ecotoxicological information

Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 28 to June 15, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Details for read across approach are included into the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
adopted on April 4th, 1984
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: three concentrations, i.e. 56, 100 and 180 mg/l and the blank, duplicate samples were taken for analysis.
- Sampling frequency: at 48 hrs from the Fresh media after renewal and at the end of the test (at 96 hrs); duplicate samples at 48 hrs just before renewal of the test media were taken only from 180 mg/l to confirm that the initial series of test concentrations was correct.
- Volume: 10 ml in duplicate and extra samples of 50 ml for additional analysis if necessary, from the approximate centre of the test vessels.
- Sample storage conditions before analysis: the samples were stored at -20 °C.
Vehicle:
no
Details on test solutions:
The test media were prepared using stock solutions in distilled water. For this purpose the test substance was added quantitatively providing stocks of 2000 mg/l and 5000 mg/l respectively.
At the renewal at 48 hours only one stock was prepared of 2000 mg/l.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: carp.
- Source: Zodiac, proefacc, "De Haar Vissen", L.U. Wegeningen, the Netherlands.
- Length at study initiation: 2.2 ± 0.15 cm.
- Weight at study initiation: 0.18 ± 0.050 g.
- Characteristics: pathogen-free F1 from a single parent-pair.
- Feeding: no feeding from 24 hours prior to the test and during the total test period.

ACCLIMATION
- Acclimation period: at least 14 days after delivery.
- Acclimation conditions: following a 48-hour settiing-in period prior to testing, the fish were adapted to the test medium without test substance for at least 7 days.
- Type of food during acclimation: Trouvit or Artemia.
- Feeding frequency during acclimation: daily.
- Health during acclimation: in the batch of fish used for the test, mortality during seven days prior to the start of the test was less than 5 %.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
The temperature of the test medium measured in the blank control ranged from 21 to 21.5 °C.
pH:
The pH increased with increasing concentration, i.e. from 7.8 in the blank to 9.0 at the highest concentration. In the blank and the test solutions that remained during the total exposure period, pH ranged from 7.8 to 8.5.
Dissolved oxygen:
Oxygen concentration in the test media was found to be > 5 mg/l for all measurements performed during the final study.
Nominal and measured concentrations:
56, 100, 180, 320, 560 and 1000 mg/l (nominal).
Details on test conditions:
TEST SYSTEM
- Test vessel: 10 litres, all-glass.
- Renewal rate of test solution: after 48 hours.
- No. of organisms per vessel: 10 fish per concentration and control.
- No. of vessels per concentration: one replicate.
- No. of vessels per control: one replicate.
- Biomass loading rate: 0.6 g fish/litre, i.e. 10 fish per 3 litres of test medium.

TEST MEDIUM / WATER PARAMETERS
- Preparation of test medium: dutch standard water formulated using Milli-Ro water, according to Adema (Adema D.M.M. 1980).
- Dilution water: tap-water purified by reverse osmosis.
- Ca2+: 54.4 mg/l
- Mg2+: 17.5 mg/l
- Na+: 27.4 mg/l
- K+: 7.8 mg/l
- Cl-: 96.6 mg/l
- Sulphate: 70.1 mg/l
- Bicarbonate: 84.8 mg/l
- Hardness: after aeration the hardness was 11.8 °DH.
- pH: 8.2 ± 0.2

OTHER TEST CONDITIONS
- Photoperiod: 16 hours daily.

MEASUREMENTS
- Mortality and other effects: at 1.5, 24, 48, 72 and 96 hours following the start of exposure. Dead fish were removed when observed. No effects other than mortality could be scored owing to the dark colour of the test concentrations. Therefore effects were scored only at the end of the test.
- Fish length and weight: a representative number (at least 10) of the batch of fish, from which fish were used for the test, was weighed and measured prior to the start of the test.
- Dissolved oxygen content: daily in all vessels, beginning at the start of the test.
- Temperature of medium: daily in one control vessel, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes
- Number of fish per concetration: 5 fish.
- Test concentrations: range 0.1 to 1000 mg/l.

ACCEPTABILITY OF THE TEST
The mortality in the blank and, if relevant, in the control containing the vehicle should not exceed 10 %.
Reference substance (positive control):
yes
Remarks:
a reference test with pentachlorophenol (PCP) is carried out every 3 months
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
180 - 320 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
After 24 hours of exposure, all fish had died at nominally 320 mg/l and higher. After 96 hours of exposure the lowest concentration at which 100 % mortality was recorded was 320 mg/l, while 20 % mortality was recorded at 180 mg/l. No mortality of fish was observed at and below 100 mg/l.
No effects other than mortality could be observed during the test owing to the dark colour of the test concentrations. Therefore scoring of effects was done only at the end of the test.

CONTROL
In the control, no mortality was observed.

RANGE-FINDING TEST
In the range finding, 100 % mortality of fish was recorded at 1000 mg/l and 20 % at 100 mg/l. No mortality was seen at 10 mg/l and lower.

MEASURED CONCENTRATIONS
Analysis of the sample taken from 180 mg/l just before renewal of the test media revealed that the actual test concentration was in agreement with the nominal concentration.
Analysis of the samples taken from the fresh media just after renewal of the test media (at 48 hrs) of the final test revealed that the actual concentrations were all approximately 100 % relative to the nominal concentrations. Furthermore, no significant decrease in actual concentration was recorded in the samples taken at the end of the study.
Because also no significant difference could be observed between spectra of samples taken during the second half of the exposure period (at 48 hrs and at 96 hrs, after renewal), the actual test concentration may be considered to have been stable during the 48-hour period.
Results with reference substance (positive control):
The LC50 (96h) resulted between 0.18 and 0.32 mg/l. The historicai range of the LC50 (96h) for carp recorded in reference tests approximateiy every 3 months since April 1988 is between 0.18 and 1.0 mg/l.
Sublethal observations / clinical signs:

Incidence and total mortality in the final study

Nominal conc. (mg/l) N. exposed no. dead fish total mortality (%)
1.5 hrs 24 hrs 48 hrs 72 hrs 96 hrs
blank 10 0 0 0 0 0 0
56 10 0 0 0 0 0 0
100 10 0 0 0 0 0 0
180 10 0 0 1 0 1 20
320 10 0 10 - - - 100
560 10 0 10 - - - 100
1000 10 0 10 - - - 100

Measured concentrations

Time of sampling (hrs) Concentration (mg/l) % nominal
nominal analyzed
48 (old medium) 180 173 96
49 (fresh medium) 56 56.7 101
100 92.9 / 92.9** 93 / 93
180 179 / 183** 99 / 102
96 (old medium) 56 59.1 / 58.4** 106 / 104
100 95.0 / 94.3** 95 / 94
180 182 / 181** 101 / 101

**Results of duplicate samples.

Test conditions

Nominal conc. (mg/l) pH values
0 hrs 24 hrs 48 hrs
old
48 hrs
renewal
72 hrs 96 hrs
blank 7.8 8.2 8.2 8.2 8.2 8.0
56 8.2 8.3 8.2 8.2 8.0 8.1
100 8.3 8.4 8.3 8.2 8.0 8.1
180 8.5 8.4 8.3 8.2 8.1 8.1
320 8.7 - - - - -
560 8.8 - - - - -
1000 9.0 - - - - -
Oxygen (mg/l)
blank 8.7 8.8 8.7 8.6 8.9 9.4
56 8.6 8.7 8.7 8.6 9.0 9.4
100 8.6 8.5 8.7 8.6 9.0 9.5
180 8.7 8.5 8.6 8.6 8.8 9.5
320 8.7 - - - - -
560 8.6 - - - - -
1000 8.7 - - - - -

Incidence of mortality observed in reference substance pentachlorophenol

Nominal conc. (mg/l) no. exposed no. of dead fish
3.5 hrs 24 hrs 48 hrs 72 hrs 96 hrs
blank 5 0 0 0 0 0
0.18 5 0 0 1 0 0
0.32 5 0 2 2 0 1
0.56 5 0 5 - - -
1.0 5 0 5 - - -
1.8 5 4 1 - - -
Validity criteria fulfilled:
yes
Conclusions:
LC50 (96h): 180 - 320 mg/l (nominal)
Executive summary:

96-hour acute toxicity study in the Cyprinus carpio with test item was conducted according to the OECD guideline 203.

Ten fish per concentration were exposed for 96 hours in a semi-static system to a concentration range of nominally 56 to 1000 mg/l forming a geometric progression with a factor of 1.8. The test media were renewed after 48 hours.

After 24 hours of exposure all fish died at nominally 320 mg/l and higher. After 96 hours of exposure, the lowest concentration at which 100 % mortality was recorded was 320 mg/l, while 20 % mortality was recorded at 180 mg/l. No mortality of fish was observed at and below nominally 100 mg/l.

Analysis of the samples taken from 56, 100 and 180 mg/l from the fresh media just after renewal of the test media revealed actual concentrations that were all approximately 100 % relative to the nominal concentrations. Furthermore, no significant difference between spectra of these samples and those taken at the end of the study could be observed. Hence, the test substance may be considered to have been stable during the 48-hour period of renewal.

The 96h-LC50 for carp exposed to test item was between 180 and 320 mg/l with 0 % mortality at 100 mg/l and 100 % mortality at 320 mg/l.

Conclusion

LC50 (96h): 180 - 320 mg/l (nominal)

Description of key information

LC50 (96h): 180 - 320 mg/l (nominal)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
180 mg/L

Additional information

No studies on the short-term toxicity to fish are available for the Direct Blue 267. Nevertheless, a study conducted on a structural analogue, Similar Substance 01, is available; the read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included in IUCLID section 13).

96-hour acute toxicity study in the carp with Similar Substance 01 was conducted according to the OECD guideline 203. Ten fish per concentration were exposed for 96 hours in a semi-static system to a concentration range of nominally 56 to 1000 mg/l forming a geometric progression with a factor of 1.8. The test media were renewed after 48 hours. After 24 hours of exposure all fish had died at nominally 320 mg/l and higher. After 96 hours of exposure the lowest concentration at which 100 % mortality was recorded was 320 mg/l, while 20 % mortality was recorded at 180 mg/l. No mortality of fish was observed at and below nominally 100 mg/l.

Analysis of the samples taken from 56, 100 and 180 mg/l from the fresh media just after renewal of the test media revealed actual concentrations that were all approximately 100 % relative to the nominal concentrations. Furthermore, no significant difference between spectra of these samples and those taken at the end of the study could be observed. Hence, the test substance may be considered to have been stable during the 48-hour period of renewal (Bogers, 1990).