Registration Dossier

Diss Factsheets

Ecotoxicological information

Short-term toxicity to fish

Currently viewing:

Administrative data

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:
15 Jan 2018 - 20 Feb 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
GLP compliance:
yes
Specific details on test material used for the study:
Purity correction factor used: 1.32 (for the water content)
Solubility in water: >770 g/L (not corrected for water content)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and limit concentration (100 mg/L)
- Sampling method: 2.0 mL at t=0 h and t=96 h
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to the test medium. Preparation of test solutions started with the highest concentration of 100 mg/L (concentration corrected for the water content). No special treatment other than a short magnetic stirring period was needed to completely dissolve the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Strain: Linnaeus 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Length at study initiation: 3.6 ± 0.5 cm
- Weight at study initiation: 0.60 ± 0.22 g
- Method of breeding: F1 from a single parent-pair bred in UV-treated water.

HOLDING
- Acclimation period: at least 12 days after delivery
- Acclimation conditions (same as test or not): yes
- Water quality parameters were kept within the optimum limits for the respective fish species.
- Type and amount of food during acclimation: Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands)
- Health during acclimation (any mortality observed): In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
180 mg CaCO3/L
Test temperature:
20.8-21.4 °C
pH:
7.3-8.0
Dissolved oxygen:
Day 0: 9.3-9.5 mg/L
Day 1: 6.6-6.9 mg/L (morning measurement). 6.0-6.2 mg/L (afternoon measurement). Aeration was introduced after the afternoon measurement.
Day 2: 8.3-8.4 mg/L
Day 3: 8.7 mg/L
Day 4: 9.0 mg/L
Nominal and measured concentrations:
Nominal: Control and 100 mg/L (regulatory limit concentration)
Measured: 106-116 mg/L (based on Triethanolamine) and 67-74 mg/L (based on citric acid). Taking the QC recovery into account it was considered that the true concentration of the citric acid in samples approximated the nominal concentration, i.e. were at 91-83 mg/L after correction. The measured exposure concentration at 100 mg/L can be considered to be in agreement with nominal. The effect parameters are based on analytically confirmed nominal concentrations (concentration corrected for the water content and representing the pure test item). See Table 1 and 2 in 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 10 litres, all-glass, containing 8 litres of test solution
- Aeration: Aeration was introduced after ~32 hours of exposure
- No. of organisms per vessel: 7 fish per vessel
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.53 g fish/L (i.e. 7 fish per 8 litres of test medium)
- No feeding from 24 h prior to the test and during the total test period.
- Introduction of fish: Within 5 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 1.0 unit and 1.0°C.
- Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using tap water purified by reverse osmosis, with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Dissolved oxygen content, pH and temperature: Daily in all vessels with surviving fish, beginning at the start of the test (day 0).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h photoperiod daily

EFFECT PARAMETERS MEASURED
- Mortality and other effects: At 2¾, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish.

TEST CONCENTRATIONS
- Range finding study test concentrations: 0.1, 1.0, 10, 100 mg/L.
- Results used to determine the conditions for the definitive study: yes; the LC50 was expected to exceed 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol (performed Feb 2017)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- No mortality or clinical effects were observed in either of the two groups tested.
- Analyses of the cationic component showed that the measured concentration was in agreement with nominal (106-116%) throughout the test. Analyses of the anionic component showed a slightly lower recovery of 67-74% relative to nominal. However, the result obtained for the analytical Quality Control (QC) sample of 100 mg/L also showed a relatively low recovery of 81%. Taking the QC recovery into account it was considered that the true concentration of the samples approximated the nominal concentration, i.e. were at 91-83 mg/L after correction. The deviation from the nominal concentration was considered mostly due to the analytical system. It was concluded that analyses sufficiently proved that the measured exposure concentration at 100 mg/L was in agreement with nominal.
- All test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation). Aeration was introduced on the 2nd day of exposure, as the oxygen concentration tended to drop below the optimum level for testing with carp, i.e. below 5 mg/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- 96h-LC50: 0.32 mg/L (95% confidence interval: 0.22-0.46 mg/L)
- The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.
Sublethal observations / clinical signs:

Table 1: Final Test: Test Samples based on Triethanolamine

Time of sampling
[hours]

Date of sampling

Date of
analysis (a)

Concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal (b)

Analyzed

0

29 Jan 2018

12 Feb 2018

0

n.d.

n.a.

 

 

 

 

100

106

106

 

96

02 Feb 2018

12 Feb 2018

0

n.d.

n.a.

n.a.

 

 

 

100

116

116

109

(a) Samples were stored in the freezer (≤ -15°C) until the day of analysis.

(b) Corrected for the purity (i.e. 1.32)

n.d. Not detected.

n.a. Not applicable.

Table 2: Final Test: Test Samples based on Citric acid

Time of sampling
[hours]

Date of sampling

Date of
analysis (a)

Concentration
[mg/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal (b)

Analyzed

0

29 Jan 2018

20 Feb 2018

0

 0.37 (c)

n.a.

 

 

 

 

100

73.8

74

 

96

02 Feb 2018

20 Feb 2018

0

 0.37 (c)

n.a.

n.a.

 

 

 

100

67.1

67

91

(a) Samples were stored in the freezer (≤ -15°C) until the day of analysis.

(b)  Corrected for the purity (i.e. 1.32)

(c)  Estimated value, calculated by extrapolation of the calibration curve. This was considered an analytical artefact, as analytical blank injections showed comparable responses. 

n.d. Not detected.

n.a. Not applicable.

Table 3: Incidence of mortality and total mortality during the limit test

Test substance concentration (mg/L)

Initial number of fish

Cumulative mortality

Total mortality (%)

2 ¾h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

100

7

0

0

0

0

0

0

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 96h-LC50 exceeded 100 mg/L based on analytically confirmed nominal concentrations (concentration corrected for the water content and representing the pure test item).

Description of key information

The 96-h LC50 value is >100 mg/L in species (Cyprinus carpio).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

The short-term toxicity to freshwater fish was determined in a study according to OECD TG 203 and in compliance with GLP criteria. In this study, groups of 7 carp (C. carpio) were exposed for 96 hours to 0 mg/L (negative control) and a nominal limit concentration of 100 mg/L under static conditions (Charles River Laboratories, 2018). Analytical confirmation of the nominal test concentration was based on the cationic and anionic components of the test substance. Analyses of the cationic component showed that the measured concentration was in agreement with nominal (106-116%) throughout the test. Significant deviation from the nominal concentration was observed in the analysis of the anionic component, however, this deviation was considered to be mostly due to the analytical system. It was thus concluded that analyses sufficiently proved that the measured exposure concentration at 100 mg/L was in agreement with nominal. Incidences of mortality and sub-lethal effects were recorded after , 24, 48, 72 and 96 hours exposure. All OECD validity criteria were met. No mortality or clinical effects were observed in either of the two groups tested after 96 hours. Based on these findings, the 96 -h LC50 was determined to be >100 mg/L.