p-benzoquinone dioxime

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30-03-2018 to 24-08-2018

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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 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: Solutions containing 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L: controls; Test medium without test item or other additives. Spacing factor determined from range-finding test and physico-chemical properties of the substance.
- Sampling method: Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule:
at t=0 h and t=48 h, 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). Immediately after preparation, all test solutions were individually checked for Tyndall effect using a laser pen. No light scattering was observed which indicates that only the dissolved part of the test item was present in the test solutions.
- 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 brown colour. Colouration increased with increasing test item concentration. Turbidity testing indicated absence of undissolved test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Justification for species other than prescribed by test guideline: Not applicable.
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): More than 2 weeks old.
- Feeding during test: No.
- Food type: Not applicable. During breeding (pre-test): Daily, a suspension of fresh water algae.
- Amount: Not applicable.
- Frequency: Not applicable.

ACCLIMATION
- Acclimation period: No.
- Acclimation conditions (same as test or not): Not applicable.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): Not applicable.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Not applicable.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

In accordance with OECD TG 202

Hardness:

The adjusted-ISO medium M7 hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 19.0 and 20.0°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

Test conditions remained within the limits prescribed by the guideline; pH: 6-9 not varying by 1.5 units the end of the test. Actual pH: 7.9 - 8.3

Dissolved oxygen:

Test conditions remained within the limits prescribed by the guideline; oxygen:>3 mg/L at the end of the test. Actual oxygen: start: 8.6 ± 0.1 mg/L and end: 9.2 ± 0.1 mg/L

Nominal and measured concentrations:

Combined range-finding / limit test: nominal concentrations: 0 (control), 1.0, 10.0 and 100.0 mg/L
Definitive test: nominal concentrations: nominal concentrations: Solutions containing 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L: controls; Test medium without test item or other additives. Analysis of the samples taken during the final test showed that measured concentrations were in agreement with nominal throughout the exposure period (i.e. 90-100% of nominal) except for solutions at 5.6 mg/L, which was measured to be 6.4 mg/L (i.e. 120% relative to initial).
Therefore results were based on geometric mean concentrations, specifically:
Definitive test: 0 (control), 0.906, 2.2, 2.3, 5.6 and 9.2 mg/L geometric mean concentrations, respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL
- Type (delete if not applicable): all glass, open ; static.
- Material, size, headspace, fill volume: Glass, headspace: 10 mL, fill volume: 50 mL
- Aeration: No aeration of the test solutions.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel containing 50 mL of test solution
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse Osmosis (RO-water, GEON Waterbehandeling, The Netherlands); used to prepare adjusted-ISO Medium M7. The following macro salts were diluted in RO water and added to ISO Medium M7: Macro salts: CaCl2.2H2O: 211.5 mg/L ; MgSO4.7H2O: 88.8 mg/L ; NaHCO3 46.7 mg/L ; KCl: 4.2 mg/L
- Culture medium different from test medium: No, adjusted-ISO Medium M7, renewed by 50% twice per week.
- Intervals of water quality measurement: 0 hours (start) and 48 hours (end) of test for pH, and oxygen concentration. Temperature was monitored continuously.

OTHER TEST CONDITIONS
- Adjustment of pH: Not applicable.
- Photoperiod: 16 hours (light) / 8 hours (dark)
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility (including mortality), 24 hours and at 48 hours. Any other unusual behaviour was also recorded.

VEHICLE CONTROL PERFORMED: Not applicable.

RANGE-FINDING STUDY
- Test concentrations: Combined range-finding / limit test: nominal concentrations: 0 (control), 1.0, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: Yes. No immobility was observed in the control and at the lowest test concentration throughout the
exposure period. At the end of the test, complete immobility was observed at the solutions prepared at 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

7.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 6.2 - 8.9 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 3.4 - 3.7 mg/L

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations: None.
- Mortality of control: No mortalities.
- Other adverse effects control: None.
- Abnormal responses: None.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: None. Acute immobilisation observed only.
- Relevant effect levels: See below.
- Dose-response test: Yes.
- EC50/LC50: 24h-EC50 was 0.80 (C.I. 0.71 – 0.90) mg/L. 48h-EC50 was 0.33 (C.I. 0.29 – 0.38) mg/L
- Other: The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of test organism was in agreement with the historical data.

Reported statistics and error estimates:

The EC50 could not be determined using a regression method. Instead, the 24 and 48h-EC50-values were calculated by applying the Spearman-Karber procedure (non-linear; without trimming) on the percentages of affected daphnids and the logarithms of the corresponding average exposure concentrations.
Software package: ToxRat Professional v 3.2.1

Table 1.0 : pH and oxygen (mg/L) concentrations during the final test

Test Item/ Geometric average exposure conc. (mg/L)	Start (t=0 h)		End (t=48 h)
	pH	O2	pH	O2
Control	8.0	8.5	8.3	9.2
0.96	8.0	8.6	8.2	9.2
2.2	8.0	8.6	8.2	9.3
2.3	7.9	8.6	8.2	9.2
5.6	7.9	8.5	8.2	9.3
9.2	8.0	8.5	8.1	9.2

 

Table 2.0 : Number of introduced daphnids and incidence of immobility in the second combined limit/range-finding test

Time (h)	Replicate	Test Item ; nominal conc. (mg/L)
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	1	5#
	B	0	0	1	5
	C	0			5
	D	0			5
	Total immobilised	0	0	2	20
	Effect %	0	0	20	100
48	A	0	0	5	5
	B	0	0	5	5
	C	0			5
	D	0			5
	Total immobilised	0	0	10	20
	Effect %	0	0	100	100

# - Microscopic observation revealed no test item attached to the daphnids.

 

Table 3.0 : Number of introduced daphnids and incidence of immobility in the final test

Time (h)	Replicate	Test Item; Geometric average exposure conc. (mg/L) [Nominal concentration (mg/L)]
		0 (Control) [0 (Control)]	0.96 [1.0]	2.2 [1.8]	2.3 [3.2]	5.6 [5.6]	9.2 [10]
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	2	4#
	B	0	0	0	0	1	3
	C	0	0	0	0	1	3
	D	0	0	0	0	1	2
	Total immobilised	0	0	0	0	5	12
	Effect %	0	0	0	0	25	60
48	A	0	0	0	0	5	5
	B	0	0	0	1	5	5
	C	0	0	0	0	5	5
	D	0	0	0	0	5	5
	Total immobilised	0	0	0	1	20	20
	Effect %	0	0	0	5	100	100

Where: nominal concentration is enclosed in [ ] brackets.

# - Microscopic observation revealed no test item attached to the daphnids.

Validity criteria fulfilled:

yes

Conclusions:

The test item 48h-EC50 was 3.5 mg/L (C.I: 3.4 – 3.7 mg/L) based on geometric mean measured concentrations.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test under GLP. A limit test was combined with a range-finding test using static conditions was initially performed. Twenty Daphnia magna per test group (4 replicates, 5 daphnia per replicate vessel) were exposed to untreated control medium and to a test item concentration of 1.0, 10.0 and 100 mg/L. No immobilisation was seen at 1.0 mg/L whereas 100% immobilisation was observed at 10 to 100 mg/L at the 48-hour time point. A final test was performed based on the results of a finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to nominal concentration: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Analysis of the samples taken at the start of the final test showed equivalent measured concentrations of 0 (control), 1.0, 2.2, 2.3, 4.9 and 9.6 mg/L. Initially measured concentrations remained stable throughout the exposure period (90 – 100% relative to initial), except for the solution prepared at 5.6 mg/L which was measured to be 6.4 mg/L (i.e. 129% relative to initial) at the end of the test. Based on these results, the geometric mean measured exposure concentrations were calculated to be 0.96, 2.2, 2.3, 5.6, and 9.2 mg/L, respectively. No biologically relevant immobility (i.e. >10%) was observed in the control and at the three lowest test concentrations throughout the test. Complete immobility was observed at the two highest test concentrations at the end of the test. The study met the acceptability criteria. The 48h-EC50 was 3.5 mg/L (C.I: 3.4 – 3.7 mg/L) based on geometric mean measured concentrations.

Description of key information

EC50-48h (invertebrates) = 3.5 mg/L (95% CL: 3.4 – 3.7 mg/L) based on based on geometric mean measured concentrations, 48-hour, freshwater, OECD TG 202, 2018

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

3.5 mg/L

Additional information

Key study: OECD TG 202, 2018 : The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test under GLP. A limit test was combined with a range-finding test using static conditions was initially performed. Twenty Daphnia magna per test group (4 replicates, 5 daphnia per replicate vessel) were exposed to untreated control medium and to a test item concentration of 1.0, 10.0 and 100 mg/L. No immobilisation was seen at 1.0 mg/L whereas 100% immobilisation was observed at 10 to 100 mg/L at the 48-hour time point. A final test was performed based on the results of a finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to nominal concentration: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Analysis of the samples taken at the start of the final test showed equivalent measured concentrations of 0 (control), 1.0, 2.2, 2.3, 4.9 and 9.6 mg/L. Initially measured concentrations remained stable throughout the exposure period (90 – 100% relative to initial), except for the solution prepared at 5.6 mg/L which was measured to be 6.4 mg/L (i.e. 129% relative to initial) at the end of the test. Based on these results, the geometric mean measured exposure concentrations were calculated to be 0.96, 2.2, 2.3, 5.6, and 9.2 mg/L, respectively. No biologically relevant immobility (i.e. >10%) was observed in the control and at the three lowest test concentrations throughout the test. Complete immobility was observed at the two highest test concentrations at the end of the test. The study met the acceptability criteria. The 48h-EC50 was 3.5 mg/L (C.I: 3.4 – 3.7 mg/L) based on geometric mean measured concentrations.