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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Principles of method if other than guideline:
Most test fish had a somewhat larger size than the one recommended in the test guideline for the zebra fish and the loading (g fish / litre test medium) in the control and the test concentrations slightly exceeded the recommendation of the guideline (1 g/l). These deviations from the recommendations of the guideline are not regarded to be relevant to the results.
GLP compliance:
yes
Specific details on test material used for the study:
Batch number: 0900250090
Date of receipt: 2017-02-06
Expiry date: 2018-01-20
Purity: 98.87 %
Water solubility: 7.398 g/L at 20 °C
Analytical monitoring:
yes
Remarks:
HPLC
Details on sampling:
Stability of test concentration/s during exposure: Examined several times by chemical analysis (HPLC) in the freshly prepared media and in the media after 24 hours of exposure.
Storage: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.
Vehicle:
no
Details on test solutions:
Pre-treatment of test item and preparation of test item concentrations
100 mg of the test item were added each day to 1 litre of dilution water, treated for 60 seconds at 8000 rpm with an ultra turrax and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 - 12 µm and an aseptic filter (Sartobran sterile capsules) with a pore size of 0.45 µm + 0.2 µm.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 5000 mL. Finally 7 fish were given to all test item concentrations and the control.

Standard Solutions used for Calibration of the test item
Resorcindiacetate
22.8 mg of the test item were dissolved and made up with acetonitrile to the mark in a 100 mL volumetric flask to prepare a stock solution of 228 mg/L. Defined volumes of this stock solution were further diluted with acetonitrile / Millipore water (1/1) to obtain standard solutions in the range of 0.023 to 2.280 mg/L.
The concentration of the lowest used calibration solution was employed as the limit of quantification.
Resorcin
22.6 mg of the analytical standard were dissolved and made up with acetonitrile to the mark in a 100 mL volumetric flask to prepare a stock solution of 225,774 mg/L. The purity of the analytical standard was considered. Defined volumes of this stock solution were diluted with acetonitrile / Millipore water (15 %/85 %) to obtain standard solutions in the range of 0.023 to
2.258 mg/L.
The concentration of the lowest used calibration solution was employed as the limit of quantification.
These solutions were used to calibrate the HPLC-system.

Standard Solutions used for Verification of the Calibration of the test item, analytical standard and Blank Test
Resorcindiacetate
20.7 mg of the test item were dissolved and made up with acetonitrile to the mark in a 100 mL volumetric flask to prepare a stock solution of 207 mg/L. This solution was further diluted with acetonitrile / Millipore water to obtain a concentration of 0.828 mg/L. The daily verification of the calibration was performed with the diluted Standard Solution of Verification.
Resorcin
22.3 mg (respectively 24.6 mg) of the analytical standard were dissolved and made up with acetonitrile to the mark in a 100 mL volumetric flask to prepare a stock solution of 222.777 mg/L (respectively 245.754 mg/L). The purity of the analytical standard was considered. This solution was further diluted with acetonitrile / Millipore water to obtain a concentration of 0.8911 mg/L (respectively 0.9830 mg/L). The daily verification of the calibration was performed with the diluted Standard Solution of Verification.
The mean recovery rate was calculated.
Acetonitrile / Millipore water (1/1) without the test item was analysed (analytical blank).

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Source: Interaquaristik.de Shop (Germany)
Date supplied: 2017-07-04
Acclimatisation: Stock held since 2017-07-04 and acclimatised to the test conditions since then.
Temperature: 20 - 24 °C
Dissolved oxygen: > 5 mg/L
Feeding: Commercial fish food, daily. Feeding discontinued 24 h prior to test start.
Mortalities during acclimatisation period: < 5 %
Medication: none
Mean standard length (n = 42): 3.84 cm (S.D. = 0.24 cm)

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
13.6 °dH (= 243 mg/L CaCO3)
Test temperature:
22.1 to 22.8 °C
pH:
7.3 - 7.7
Dissolved oxygen:
8.9 - 7.9 mg/L (100 - 90% saturation)
Nominal and measured concentrations:
0.31, 0.63, 1.3, 2.5 and 5 mg/L (nominal)
Details on test conditions:
Exposure conditions

Test vessels: glass aquaria holding 5 L of test media covered by glass plates

Experimental design: 5 test concentrations plus 1 control
7 fish per test concentration
no feeding during the exposure period
semi-static system

Method of initiation: fish were placed in prepared media

Loading: 1.25 g body weight (wet weight) per litre

Photoperiod: 16 h light: 8 h dark

Temperature: 22.1 to 22.8 °C

Aeration: gentle aeration via narrow glass tubes

Method of administration: stock solution

Medium renewal: daily

Criteria of effects: The criterion of death used in this study was the absence of response to physical stimulation. In addition to observations on mortality at 2, 24, 48, 72 and 96 hours, type and incidence of sub-lethal effects compared with control fish were observed.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.588 - < 1.212 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
2.604 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
0.588 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1.185 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC100
Effect conc.:
2.604 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC0
Effect conc.:
0.588 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 1.212 - < 2.604 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC100
Effect conc.:
2.604 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC0
Effect conc.:
0.588 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
1.729 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limit: 1.380 - 2.245
Duration:
24 h
Dose descriptor:
LC100
Effect conc.:
5.413 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limit: 1.380 - 2.245
Duration:
24 h
Dose descriptor:
LC0
Effect conc.:
1.171 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence limit: 1.380 - 2.245
Details on results:
Criteria of effects: The criterion of death used in this study was the absence of response to physical stimulation. In addition to observations on mortality at 2, 24, 48, 72 and 96 hours, type and incidence of sub-lethal effects compared with control fish were observed.

All results are expressed in terms of geometric mean measured concentrations calculated on base of the sum of Resorcindiacetate, Resorcinmonoacetate and Resorcin. Because of a lacking dose/response relationship the LC 50 values at 48 h and 96 hours could not be determined by probit analysis.
Effective concentrations calculated as sum of Resorcindiacetate, Resorcinmonoacetate and Resorcin ranged from 106.5 % to 128.6 % of nominal values in the freshly prepared media and from 55.0 % to 121.8 % of nominal values in the media after 24 hours of exposure according to the semi-static test conditions.
Reported statistics and error estimates:
The LC 50 was calculated by probit analysis using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).

Results of the probit analysis

Results of the probit analysis: Selected effective concentrations (LCx) of the test item and their 95%- and 99%-confidence limits (according to Fieller`s theorem).

 Parameter  LC10  LC20  LC50
 Value [mg/L]  1.175  1.342  1.729
 lower 95%-cl  0.764  0.957  1.380
 upper 95%-cl  1.460  1.654  2.245
 lower 95%-cl  0.690  0.878  1.278
 upper 95%-cl  1.616  1.803  2.424

Slope function after Litchfield and Wilcoxon: 1.352

Results of the probit analysis: Selected effective concentrations (LCx) of the test item and their 95%- and 99%-confidence limits (according to Fieller`s theorem).

Parameter  LC10  LC20  LC50
 Value [mg/L]  1.012  1.068  n.d.
 lower 95%-cl  n.d.  n.d.  n.d.
 upper 95%-cl  n.d.  n.d.  n.d.
 lower 95%-cl  n.d.  n.d.  n.d.
 upper 95%-cl  n.d.  n.d.  n.d.

n.d.: not determined due to mathematical reasons or inappropriate data

Slope function after Litchfield and Wilcoxon: 1,131

Validity criteria fulfilled:
yes
Remarks:
The mortality in the controls did not exceed 10 % by the end of the test. The dissolved oxygen concentration remained above 60 % of the air-saturation value throughout the exposure period. The pH did not vary by more than 1 unit.
Conclusions:
Acute toxicity of Resorcindiacetat to Danio rerio under semi-static conditions shows a LC50 >0.588 mg/L and <1.212 mg/L after 96 hours.
Executive summary:

A study was performed to assess the acute toxicity of Resorcindiacetat to Danio rerio under semi-static conditions with daily renewal of test solutions.

The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.1 'Acute toxicity for Fish' (2008) which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 203 'Fish, Acute Toxicity Test' (1992).

Groups of seven fish of the recommended size were exposed to a range of concentrations, nominally 0.31, 0.63, 1.3, 2.5 and 5 mg/L of Resorcindiacetat dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer, a folded filter and an aseptic filter.

Observations were made on the numbers of dead fish and the incidence of sub-lethal effects after 2, 24, 48, 72 and 96 hours of exposure. The following values were determined:

 Time [h]  LC50 [mg/L]  Geometric mean LC0/LC100 [mg/L]
 2  --  3.979
 24  1.729  
 48  > 1.212 < 2.604  
 72  1.185  
 96  > 0.588 < 1.212  

All results are expressed in terms of geometric mean measured concentrations calculated on base of the sum of Resorcindiacetate, Resorcinmonoacetate and Resorcin. Because of a lacking dose/response relationship the LC 50 values at 48 h and 96 hours could not be determined by probit analysis.

Effective concentrations calculated as sum of Resorcindiacetate, Resorcinmonoacetate and Resorcin ranged from 106.5 % to 128.6 % of nominal values in the freshly prepared media and from 55.0 % to 121.8 % of nominal values in the media after 24 hours of exposure according to the semi-static test conditions.

The hardness of the dilution water used was 13.6 °dH (= 243 mg/L CaCO3).

Description of key information

A study was performed to assess the acute toxicity of Resorcindiacetat to Danio rerio under semi-static conditions with daily renewal of test solutions.

The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.1 'Acute toxicity for Fish' (2008) which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No. 203 'Fish, Acute Toxicity Test' (1992).

Groups of seven fish of the recommended size were exposed to a range of concentrations, nominally 0.31, 0.63, 1.3, 2.5 and 5 mg/L of Resorcindiacetat dissolved in dilution water. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer, a folded filter and an aseptic filter.

Observations were made on the numbers of dead fish and the incidence of sub-lethal effects after 2, 24, 48, 72 and 96 hours of exposure. The following values were determined:

 Time [h]  LC50 [mg/L]  Geometric mean LC0/LC100 [mg/L]
 2  --  3.979
 24  1.729  
 48  > 1.212 < 2.604  
 72  1.185  
 96  > 0.588 < 1.212  

All results are expressed in terms of geometric mean measured concentrations calculated on base of the sum of Resorcindiacetate, Resorcinmonoacetate and Resorcin. Because of a lacking dose/response relationship the LC 50 values at 48 h and 96 hours could not be determined by probit analysis.

Effective concentrations calculated as sum of Resorcindiacetate, Resorcinmonoacetate and Resorcin ranged from 106.5 % to 128.6 % of nominal values in the freshly prepared media and from 55.0 % to 121.8 % of nominal values in the media after 24 hours of exposure according to the semi-static test conditions.

The hardness of the dilution water used was 13.6 °dH (= 243 mg/L CaCO3).

Key value for chemical safety assessment

LC50 for freshwater fish:
0.588 mg/L

Additional information