p-benzoquinone dioxime

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:

06-04-2018 to 16-08-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
(i) test item (prior to testing): In refrigerator (2 - 8°C), container flushed with nitrogen.
(ii) test solution samples were Samples were stored in a freezer (≤-15°C) until analysis.
- Stability under test conditions: Stable (full details available in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Soluble up to 100 mg/L in water and stable (full details available in the full study report).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not applicable.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Not applicable.
- Final preparation of a solid: Not applicable.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Not applicable.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): Not applicable.

OTHER SPECIFICS: Not applicable.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Definitive test: nominal concentrations: 0 (control), 10, 18, 32, 56 and 100 mg/L
Spacing factor (1. 8) determined from range-finding test and physico-chemical properties of the substance.
Controls: test water without test item but treated in the same way as the test item solutions.
- Sampling method: Samples for analysis were taken from the control and all test concentrations at t=0h, t=24h and t=96h. An extra sample was taken at 32 mg/L after 72h exposure. Volume: 2 ml from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis (≤ -15°C). Reserve samples of 2 ml were taken for possible analysis. If not used, these samples were stored in a freezer.

Vehicle:

no

Details on test solutions:

PPREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with loading rates individually prepared in the range of 1.0 to 100 mg/L. A three-day period of magnetic stirring was applied to accelerate dissolution of the test item in test medium. Thereafter, the aqueous solutions were collected by filtration through a 0.45 μm membrane filter (RC55, Whatman, in the range-finding test and a PES450, Supor, in the final test)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None. All test solutions were clear and had a yellow to dark brown colour at the end of the preparation procedure. Coloration increased with increasing loading rate of test item. Turbidity testing indicated absence of undissolved test item.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish (Danio rerio, Teleostei, Cyprinidae)
- Strain: Not reported
- Source: Recognised supplier (listed in full study report)
- Age at study initiation (mean and range, SD): Not reported.
- Length at study initiation (length definition, mean, range and SD): RF test: mean: 2.3 ± 0.2 cm ; definitive: 2.5 ± 0.2 cm
- Weight at study initiation (mean and range, SD): mean: RF test: mean: 0.07 ± 0.03 g ; definitive: 0.12 ± 0.04 g
- Method of breeding: Not reported
- Maintenance of the brood fish: Not reported

ACCLIMATION
- Acclimation period: > 7 days (Actual: 12 days)
- Acclimation conditions (same as test or not): Yes : Adjusted ISO medium, formulated using RO-water
- Type and amount of food during acclimation: Pelleted fish food (recognised supplier).
- Feeding frequency during acclimation: Daily (feeding ceased 24 hours before test initiation).
- Health during acclimation (any mortality observed): None.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: None.

FEEDING DURING TEST
- Food type: No.
- Amount: Not applicable.
- Frequency: Not applicable.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None.

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

21-25 ºC ; constant: ± 2 ºC (actual 21 - to 22 ºC)

pH:

pH = 7.7 ± 0.3

Dissolved oxygen:

8.22-8.90 mg/L (fresh media); 5.08-6.40 mg/L (or 59.1 to 74.6% ASV) (expired media)

Salinity:

Not applicable.

Nominal and measured concentrations:

Range Finding Test:
Nominal Concentrations: 0 (control), 1.00, 10.00, 100 mg/L
Analytically confirmed nominal concentrations since measured concentration was maintained 101 – 109% throughout the test period
Definitive Test:
Nominal test concentrations: 0 (control), 10, 18, 32, 56, 100 mg/L
Analytically confirmed nominal concentrations since measured concentration was maintained 100 – 112% throughout the test period.

Details on test conditions:

TEST SYSTEM
- Test vessel: 3.5 L glass.
- Type (delete if not applicable): all glass ; open
- Material, size, headspace, fill volume: ca.3.0 L fill volume test media per test vessel
- Aeration: no
- Renewal rate of test solution (frequency): Not applicable (static)
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: < 1.0 g/L (actual 0.28 g/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO-water (equipment cited in full study report).
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: < 10 μS/cm
- Culture medium different from test medium: No. Reconstituted ISO Medium in accordance with OECD TG 203.
- Intervals of water quality measurement: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and visible abnormality recorded at least the following: 24 h, 48 h, 72 h and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8 (Definitive)
- Range finding study: yes (static)
- Test concentrations: Range Finding Test:
Nominal Concentrations: 0 (control), 1.00, 10.00, 100 mg/L (measured concentration was maintained 101 – 109%) ; Definitive Test: Nominal test concentrations: 0 (control), 10, 18, 32, 56, 100 mg/L (measured concentration was maintained 100 – 112%).
- Results used to determine the conditions for the definitive study: Yes. Static test indicated possible to maintain test item concentrations during exposure. Results: At 1 mg/L and 10 mg/L: 0% cumulative mortality at 96 h time point. At 100 mg/L: 100% cumulative mortality at 24 through 96 h time points.

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence interval: 18 - 32 mg/L ; based on analytically confirmed nominal concentrations

Details on results:

- Behavioural abnormalities: See table 1 and table 2
- Observations on body length and weight: Not reported
- Other biological observations: Not reported
- Mortality of control: 0%
- Other adverse effects control: No abnormalities observed in the control.
- Abnormal responses: Sub lethal responses are reported in table 1 and table 2
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Turbidity measurements indicated the absence of any undissolved test item. Precipitation of particulate material was observed at concentrations of 18 and 32 mg/L. As no decrease of test item concentration was measured during the test, the observed precipitate was considered most likely not test item related.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Mortality: Yes.
- LC50: 0.17 mg/L (95% CL 0.13 – 0.23 mg/L) in 96 h
- Other: In agreement with laboratory historical database

Reported statistics and error estimates:

24 and 48-hour LC50-values were determined by applying the Spearman-Karber procedure (non-linear; without trimming) on the mortality percentages and the logarithms of the corresponding test item concentrations.

The 72 and 96-hour LC50-values could not be determined using a regression method. This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred. Instead, the 72 and 96h-LC50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.

Software package: ToxRat Professional v 3.2.1.

Sublethal observations / clinical signs:

Table 1.0 : nominal, measured concentrations and observations during the definitive test

Test Item nominal conc. (mg/L)	Test Item measured conc. (mg/L) *	Initial number of fish	Cumulative mortality					Total Mortality (%)
			3h	24h	48h	72h	96h
0 (Control)	n.d.	7	0	0	0	0	0	0
10	10.4	7	0	0	0	0	0	0
18	18.7	7	0	0	0 #1	0 #1,#2	0 #1,#2	0
32	32.1	7	0	4	6 #1	7 #1,#2	7	100
56	60.3	7	0	7 #1	7	7	7	100
100	108	7	2	7	7	7	7	100

Where:

* = 96 hours, unless complete mortality when measured at that time point

#1 = faeces observed

#2 = precipitated observed

 

Table 2.0 : clinical effects observed in the final test

Test Item nominal conc. (mg/L)	Time of recording (hours)	Specification of effects	Relative number
32	48	Swimming at the surface	1/1
100	3	Darkly coloured	5/5

 

 

Validity criteria fulfilled:

yes

Conclusions:

The test item 96h-LC50 was 24 mg/L (C.I: 18 – 24 mg/L) based on analytically confirmed nominal concentrations.

Executive summary:

The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour static test according to OECD TG 203 guideline under GLP. Based on the results of a range finding test, the nominal loading rates of 0 (control), 10, 18, 32, 56 and 100 mg/L were selected for definitive testing. The total exposure period was 96 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 hours, 72 hours (single sample) and at the end of the test. No mortality or other clinical effects were observed in the control and at the two lowest test concentrations throughout the test. Analytical measurements were performed by Ultra Performance Liquid Chromatography (UPLC-TUV). Samples taken from all test concentrations and the control were analysed. The measured concentrations were at the level of nominal loading rates (i.e. 100 – 112%) throughout the test. Therefore effect levels were expressed based on analytically confirmed nominal concentrations. All relevant validity criteria were met. Under the conditions of this study, the 96h LC50 was 24 mg/L (C.I. 18 – 32 mg/L) based on analytically confirmed nominal concentrations.

Description of key information

LC50 (fish) = 24 mg/L (95% CL: 18 – 32 mg/L) based on analytically confirmed nominal concentrations, 96-hour, freshwater, OECD TG 203, 2018

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

24 mg/L

Additional information

Key data: OECD TG 203, 2018 : The acute toxicity of the test item to Zebra fish (Danio rerio) was determined in a 96 hour static test according to OECD TG 203 guideline under GLP. Based on the results of a range finding test, the nominal loading rates of 0 (control), 10, 18, 32, 56 and 100 mg/L were selected for definitive testing. The total exposure period was 96 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 hours, 72 hours (single sample) and at the end of the test. No mortality or other clinical effects were observed in the control and at the two lowest test concentrations throughout the test. Analytical measurements were performed by Ultra Performance Liquid Chromatography (UPLC-TUV). Samples taken from all test concentrations and the control were analysed. The measured concentrations were at the level of nominal loading rates (i.e. 100 – 112%) throughout the test. Therefore effect levels were expressed based on analytically confirmed nominal concentrations. All relevant validity criteria were met. Under the conditions of this study, the 96h LC50 was 24 mg/L (C.I. 18 – 32 mg/L) based on analytically confirmed nominal concentrations.