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Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 April 2018 to 23 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- The DOC in the test vessels was measured at the beginning and at the end of the study.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing 10.1 mg/L with the corresponding amount of tap water and shaking for 24 hours. The resulting solution was filtrated through 0.45 μm Cellulose ester filters.
- The lower treatments were prepared by dilution of this WAF with the corresponding amount of dilution water. This procedure was in agreement with the OECD guidance document no. 23 for the testing of mixtures which are toxic at low loading rates (≤ 1 mg/L). In a non-GLP pre-test at the loading rate 1 mg/L slight toxicity was observed.
- The individual preparation of loading rates lower than 1 mg/L is technically not possible and therefore dilution of the WAF is the applicable method.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Strain: Danio rerio HAMILTON-BUCHANAN
- Source: The animals were obtained from a commercial supplier (Dehner Garten Center, 67433 Neustadt). The fish arrived at the testing facility on 17 January 2018.
- Age at study initiation: Sexually immature young fish.
- Length at study initiation: 2 ± 1 cm

ACCLIMATION
- Acclimation period: At least 12 days under test conditions.
- Acclimation conditions:
Vessels: Polyethylene aquaria.
Medium: Chlorine-free tap water.
Photo period: 12/12 hours, using neon tubes.
Temperature: 23 ± 2 °C
- Type and amount of food during acclimation: Three times a day with warmwater fish food. 24 hours before the start of the test, the test fish were no longer fed.
- Health during acclimation: During 7 days before the test, no mortality occurred.

FEEDING DURING TEST: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

95 h

Remarks on exposure duration:

Endpoint of the test is the 96-hour LC50. The last measurements were taken after 95 hours. The test duration was therefore within the permissible range of 96 ± 1 hours.

Hardness:

0.71 mmol/L

Test temperature:

22.5 – 23.1 °C

pH:

80. - 8.2

Conductivity:

174 μS/cm (at 25 °C)

Nominal and measured concentrations:

Nominal: 0.46, 1.0, 2.2, 4.6 and 10 mg/L
Calculated concentration from the geometric mean of the measured concentration of the highest tested concentration: 0.17, 0.36, 0.79, 1.65 and 3.59 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria, maximal volume 10 L
- Material, size, headspace, fill volume: 7 L
- Aeration: Accomplished with glass tubes
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1 vessel, each containing 7 L test solution and 7 fish
- No. of vessels per control (replicates): 1 vessel, containing 7 L tap water and 7 fish
- Biomass loading rate: 1 fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Good quality drinking water was used. The most recent analysis showed the following composition: sodium 4.1 mg/L, potassium 2.5 mg/L, calcium 25 mg/L, magnesium 2.1 mg/L, aluminium 0.020 mg/L, iron 0.007 mg/L, manganese <0.005 mg/L, ammonium <0.05 mg/L, nitrate 1.8 mg/L, nitrite <0.005 mg/L, chloride 9 mg/L, sulphate 9.2 mg/L, total organic carbon (TOC) 0.6 mg/L, antimony <0.001 mg/L, arsenic <0.001 mg/L, lead <0.001 mg/L, cadmium <0.0001 mg/L, chromium <0.001 mg/L, copper 0.001 mg/L, nickel <0.001 mg/L, mercury <0.0001 mg/L, selenium <0.001 mg/L, bor <0.01 mg/L, cyanide <0.005 mg/L, fluoride <0.1 mg/L, benzene <0.1 μg/L, polycyclic aromatic hydrocarbons <0.008 μg/L, chlorinated organic compounds < 1 μg/L, pesticides and biocides < LOD. pH = 8.26. Conductivity at 25 °C = 174 μS/cm. Hardness = 0.71 mmol/L.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Every 23 to 24 hours, the pH and O2-concentration of the test solutions were measured.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 12/12 hours using neon tubes.

EFFECT PARAMETERS MEASURED:
- Observations were made every 23 to 24 hours, documenting mortalities. A fish was considered dead, if no visible movement could be observed, and if touching of the caudal peduncle produced no reaction.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC10

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

ANALYTICAL DETERMINATIONS
- Due to low solubility of test material only in the highest tested concentration (prepared WAF) test material was clearly determinable. The measured DOC concentrations in the lower concentrations were in the same, very low, range as the blank control. Since the measured DOC in the control was slightly higher than in the 3 lowest concentrated treatments, the DOC of the control was not subtracted from the DOC of the treatments.
- As the two highest concentrations showed mortality between 29 and 100 %, and the lower concentrations were prepared by dilution of the highest concentrated treatment, the test material must have been present in the test solutions. Therefore, the determination of the biological results was based on the calculated concentrations from the geometric mean of the measured concentration of the highest tested concentration with the respective dilution factor. Geometric mean is calculated by multiplication of the n participating concentrations and taking the nth root.

VALIDITY
- The mortality in the blank control may not exceed 1 fish at the end of the test. No mortality occurred in the blank control.
- The dissolved oxygen concentration must be at least 60 % throughout the test. The concentration of dissolved oxygen was at least 7.4 mg/L or 89 % throughout the test.
- The pH-value in the test solutions should not vary by more than 1 unit during the test. The highest variation was 0.2 units.

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software. The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.2.1.

Sublethal observations / clinical signs:

Table 1: Mortalities

Nominal Concentration (mg/L)	Total Number of Fish Used	Number of Dead Fish
		24 Hours	48 Hours	72 Hours	95 Hours
Blank Control	7	0	0	0	0
0.46	7	0	0	0	0
1.0	7	0	0	0	0
2.2	7	0	0	0	0
4.6	7	1	2	2	2
10	7	7	7	7	7

 

Table 2: Measured Concentrations IC and TC

Nominal Concentration (mg/L)	*Measured TC t=0 h (mg/L)	*Measured TC t=95 h (mg/L)	*Measured IC t=0 h (mg/L)	*Measured IC t=95 h (mg/L)
Blank Control	20.43	22.57	19.66	21.40
0.46	19.00	21.91	18.69	21.20
1.0	19.18	22.00	18.56	20.92
2.2	19.70	23.06	19.16	22.22
4.6	21.38	25.50	20.18	23.32
10	22.32	27.75	20.45	24.16

LOQ (Limit of quantification) TC = 4.07 mg/L

LOQ (Limit of quantification) IC = 1.34 mg/L

* Mean of two or three measurements. At least 2 measurements were performed. If the coefficient of variation was > 2 %, a third measurement was performed and the value with the largest deviation was considered an outlier.

 

Table 3: Measured Concentrations DOC

Nominal Concentration (mg/L)	Measured DOC (TI-IC) t=0 h (mg/L)	Measured DOC (TI-IC) t=95 h (mg/L)
Blank Control	0.77	1.17
0.46	0.30	0.71
1.0	0.61	1.08
2.2	0.54	0.84
4.6	1.19	2.18
10	1.87	3.58

Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results. The measured DOC in the control was slightly higher than in the 3 lowest concentrated treatments. Therefore the DOC of the control was not subtracted from the DOC in the treatments.

Table 4: Calculated Test Material Concentrations DOC

Nominal Concentration (mg/L)	*Calculated Concentration of Test Material t=0 h (mg/L)	*Calculated Concentration of Test Material t=95 h (mg/L)
Blank Control	-	-
0.46	0.42	0.98
1.0	0.85	1.50
2.2	0.76	1.17
4.6	1.65	3.03
10	2.60	4.97

* Based on an carbon content of the test material of 72.00 %.

 

Table 5: Geometric Mean

Nominal Concentration of Test Material (mg/L)	Geometric Mean of Measured Concentration (mg/L)	Calculated Concentration From the Geometric Mean of the Measured Concentration of the Highest Tested Concentration (mg/L)
Blank Control	-	-
0.46	0.64	0.17
1.0	1.13	0.36
2.2	0.94	0.79
4.6	2.24	1.65
10	3.59	3.59

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the 96 hour LC50 of the test material was 1.8 mg/L.

Executive summary:

The short-term toxicity of the test material to fish was investigated in accordance with the standardised guidelines OECD 203 and EU Method C.1., under GLP conditions using Danio rerio.

One valid experiment was performed. The acute toxicity against Danio rerio was tested using a static test design. The study was performed using five concentrations ranging from 0.46 to 10 mg/L nominal concentration. For each test concentration and the blank control, 7 fish were exposed to the test material for 96 hours in a static test system. After 24, 48, 72 and 95 hours, the number of dead fish was counted.

The two highest concentrations showed mortality between 29 and 100 %.

The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing 10 mg/L with the corresponding amount of tap water and shaking for 24 hours. The resulting solution was filtrated through 0.45 μm cellulose ester filters. Lower test concentrations were prepared by dilution of the stock WAF (10 mg/L) in tap water. This was in accordance with the OECD Guidance Document No. 23 where it is stated that dilution of a stock WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.

At the start and at the end of the test, the content of the test material in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

Due to low solubility of test material only in the highest tested concentration (prepared WAF) test material was clearly determinable. The measured concentrations in the lower concentrations were in the same, very low, range as the blank control. Since the measured DOC in the control was slightly higher than in the 3 lowest concentrated treatments, the DOC of the control was not subtracted from the DOC of the treatments.

As the two highest concentrations showed mortality between 29 and 100 %, and the lower concentrations were prepared by dilution of the highest concentrated treatment, the test material must have been present in the test solutions. Therefore, the determination of the biological results was based on the calculated concentrations from the geometric mean of the measured concentration of the highest tested concentration with the respective dilution factor.

Under the conditions of this study the 96 hour LC50 of the test material was 1.8 mg/L.

Description of key information

Under the conditions of this study the 96 hour LC50 of the test material was 1.8 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.8 mg/L

Additional information

The short-term toxicity of the test material to fish was investigated in accordance with the standardised guidelines OECD 203 and EU Method C.1., under GLP conditions using Danio rerio. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

One valid experiment was performed. The acute toxicity against Danio rerio was tested using a static test design. The study was performed using five concentrations ranging from 0.46 to 10 mg/L nominal concentration. For each test concentration and the blank control, 7 fish were exposed to the test material for 96 hours in a static test system. After 24, 48, 72 and 95 hours, the number of dead fish was counted.

The two highest concentrations showed mortality between 29 and 100 %.

The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing 10 mg/L with the corresponding amount of tap water and shaking for 24 hours. The resulting solution was filtrated through 0.45 μm cellulose ester filters. Lower test concentrations were prepared by dilution of the stock WAF (10 mg/L) in tap water. This was in accordance with the OECD Guidance Document No. 23 where it is stated that dilution of a stock WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.

At the start and at the end of the test, the content of the test material in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

Due to low solubility of test material only in the highest tested concentration (prepared WAF) test material was clearly determinable. The measured concentrations in the lower concentrations were in the same, very low, range as the blank control. Since the measured DOC in the control was slightly higher than in the 3 lowest concentrated treatments, the DOC of the control was not subtracted from the DOC of the treatments.

As the two highest concentrations showed mortality between 29 and 100 %, and the lower concentrations were prepared by dilution of the highest concentrated treatment, the test material must have been present in the test solutions. Therefore, the determination of the bio-logical results was based on the calculated concentrations from the geometric mean of the measured concentration of the highest tested concentration with the respective dilution factor.

Under the conditions of this study the 96 hour LC50 of the test material was 1.8 mg/L.