p-benzoquinone dioxime

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-03-2018 to 03-09-2018

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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), 0.1, 0.32, 1.0, 3.20 and 10 mg/L: controls; Test medium without test item or other additives. Spacing factor determined from combined limit/range-finding test and physico-chemical properties of the substance. Singular samples for possible analysis were taken from all test concentrations and the control; Frequency at t=0 h, t=24 h and t=72 h; further maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
- Sampling method: 2.0 mL from the approximate centre of the test vessels; At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- 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. Test solutions at the end of the preparation procedure, the solution prepared at 0.10 mg/L was clear and colourless. All other test solutions were clear and had a yellow colour. Coloration increased with increasing test item concentration. Turbidity testing indicated absence of undissolved test item.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days (in pre-culture under the same conditions as the test).
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

ACCLIMATION
- Acclimation period: 3 - 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): No.
Stock Culture Medium M1 ; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis) with the following composition: NaNO3 500 mg/L; K2HPO4.3H2O 52 mg/L; MgSO4.7H2O 75 mg/L; Na2CO3.10H2O 54 mg/L; C6H8O7.H2O 6 mg/L; NH4NO3 330 mg/L; CaCl2.2H2O 35 mg/L; C6H5FeO7.xH2O 6 mg/L; H3BO3 2.9 mg/L; MnCl2.4H2O 1.81 mg/L; ZnCl2 0.11 mg/L; CuSO4.5H2O 0.08 mg/L; (NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-Culture and definitive test adjusted-Medium M2 : NH4Cl 15 mg/L; MgCl2.6H2O 12 mg/L; CaCl2.2H2O 18 mg/L; MgSO4.7H2O 15 mg/L; KH2PO 1.6 mg/L; FeCl3.6H2O 64 µg/L; Na2EDTA.2H2O 100 µg/L; H3BO3 185 µg/L; MnCl2.4H2O 415 µg/L; ZnCl2 3 µg/L; CoCl2.6HO 1.5 µg/L; CuCl2.2H2O 0.01 µg/L; Na2MoO4.2H2O 7 µg/L; NaHCO3 300 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L); pH 8.1 ± 0.2
- Any deformed or abnormal cells observed: None reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

In accordance with OECD TG 201

Post exposure observation period:

None. At the end of the final test microscopic observations were performed on the solution prepared at 1.0 mg/L to observe for any abnormal appearance of the algae compared to the control.

Hardness:

Ca+Mg: 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

pH:

0 hours: pH 8.2 ± 0.1 ; 72 hours: pH 7.9 - 8.1 (definitive test concentrations) and pH 8.1 (controls). pH did not vary more than 1.5 units.

Nominal and measured concentrations:

Combined Limit/Range-finder test: 0 (control), 1.0, 10 and 100 mg/L
Final test: 0 (control), 0.1, 0.32, 1.0, 3.20 and 10 mg/L
Equivalent Time Weighted Average Concentrations: 0 (control), 0.050, 0.16, 0.74, 2.80 and 9.9 mg/L
See table 3 for nominal and measured concentrations at initial exposure and during the course of the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass.
- Type (delete if not applicable): Closed - Static
- Material, size, headspace, fill volume: Glass, 250 ml, fill volume: 30 mL test solution, closed
- Aeration: Vessel shaken continuously.
- Initial cells density: 1 x 10^4 cells/ml.
- Control end cells density: Mean (of replicates after 72 hours) 264.8 x10^4 cells/mL (increase of factor: x265)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control; 1 extra replicate of test group for sampling purposes at 24h; 1 or 2 replicates of each test concentration without algae.
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. adjusted-Medium M2.
- Detailed composition if non-standard medium was used: adjusted M2 according to OECD 201 using RO-water: NH4Cl: 15 mg/L; MgCl2.6H2O: 12 mg/L; CaCl2.2H2O: 18 mg/L; MgSO4.7H2O: 15 mg/L; KH2PO4: 1.6 mg/L; FeCl3.6H2O: 64 µg/L; Na2EDTA.2H2O: 100 µg/L; H3BO3: 185 µg/L; MnCl2.4H2O: 415 µg/L; ZnCl2: 3 µg/L; CoCl2.6H2O: 1.5 µg/L; CuCl2.2H2O: 0.01 µg/L; Na2MoO4.2H2O: 7 µg/L; NaHCO3: 300 mg/L; Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L); pH 8.1 ± 0.2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: Yes. Medium M1 used for stock culture medium. adjusted-Medium M2 used for pre-culture medium and test medium. See above for more information.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 118 to 122 µE/m2/s.
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer at 680 nm with immersion probe (pathlength = 10mm). Algal medium used as blank.
- Other: Initial cell density: Cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2. In definitive test justified from the results of the combined limit/range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to dilutions representing 1.0, 10 and 100 mg/L in the range-finding test. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval. In the definitive test 5 concentrations were used at 0 (control), 0.1, 0.32, 1.0, 3.20 and 10 mg/L.
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.0 and 1.4 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.23 - 0.26 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.15 - 0.27 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.072 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 0.065 - 0.080 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control at 10 mg/L nominal concentration.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: Not reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Yes. At low concentrations 0.050 to 0.74 mg/L TWA yield inhibition was negative between 48-72h. This was low dose stimulation and was taken into account in the data analysis and effect level calculations.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Final test: During the exposure period the measured concentrations decreased in the range finder and definitive test from nominal values. During the 72-hour exposure period, the measured concentration in the solution prepared at 0.10 mg/L had decreased below the limit of detection (LOD; 0.029 mg/L). The remaining measured concentrations were at 19-106% relative to initial. It should be noted that stability of exposure concentrations increased with increasing nominal concentrations. Measured concentrations in the solution without algae (at 1.0 mg/L) showed an unrealistic increase in exposure concentrations at the end of the test, i.e. 301% of initial. Consequently, it was decided to analyse the reserve samples, which showed a comparable result. The reason for the observed unrealistic increase was unknown. However, the concentrations measured in the samples taken from solutions with algae were comparable with concentrations measured in the samples taken from solutions without algae for the first 24 hours of exposure. Consequently, it can be stated that the presence of the algae had no effect on the test item concentration in test medium.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was 1.6 mg/L with a 95% confidence interval ranging from 1.5 to 1.6 mg/L. The historical ranges for growth rate inhibition range between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the present test system is within the historical range. The EC50 for yield inhibition (72h-EYC50) was 0.50 mg/L with a 95% confidence interval ranging from 0.48 to 0.52 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the present test system was within range.
- Other: The sensitivity of the test system was in agreement with the historical data.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller) or inhibition of yield (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

A concentration-related increase of growth rate inhibition and yield reduction was recorded at TWA concentrations of 0.16 mg/L and higher. Statistically significant inhibition of growth rate was found at TWA concentrations of 0.74 mg/L and higher. Statistically significant reduction of yield was found at TWA concentrations of 0.16 mg/L and higher.

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield reduction versus the logarithms of the corresponding TWA concentrations of the test item.
Full details of the statistical analysis are reported in the full study report.

Table 1.0 : Mean cell densities (x10^4 cells/ml) during the range-finding test

Time (h)	Test Item, nominal conc. (mg/L)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	4.9	n.d.	n.d.	6.6
48	45	n.d.	n.d.	16
72	277	20	1.0	11

n.d. = Not determined

Table 2. Percentage reduction of growth rate and inhibition of yield during the range-finding test

Test Item, nominal concentration (mg/L)	Mean	Std. Dev.	n	%Inhibition, Growth rate	Mean	Std. Dev.	n	%Inhibition
Control	1.874	0.0268	6	0.0	276.2	23.07	6	0.0
1.0	0.993	0.0211	3	47	18.7	1.22	3	93
10	0.000	0.0000	3	100	0.0	0.00	3	100
100	0.705	0.2492	6	62	9.7	9.62	6	96

 

Table 3. Measured concentrations versus nominal concentrations: final test

Test item, nominal concentration (mg/L)	Measured concentration (mg/L)					TWA (mg/L)
	t=0h	t=24h	% initial	t=72 h	% initial
0.10	0.0814	0.058	71.2	0.029 #1	35.6	0.050
0.32	0.291	0.231	79.4	0.054 #2	18.6	0.16
1.0	0.925	0.822	88.9	0.0561	6.8	0.74
1.0 #WA	0.937	0.856	91.4	2.83	302.0	1.3
3.2	2.95	2.96	100.3	2.62	88.8	2.8
10	9.41	10.0	106.3	10.0	106.3	9.9

#WA – without algae

#1 : LOD = 0.029 mg/L

#2 : calculated by extrapolation of the calibration curve

 

Table 4.Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

TWA concentration, test item (mg/L)	Mean	Std. Dev.	n	%Inhibition growth rate	Mean	Std. Dev.	n	%inhibition yield
Control	1.859	0.0146	6	0.0	263.8	11.56	6	0.0
0.050	1.876	0.0100	3	-0.88	276.9	8.41	3	-5.0
0.16	1.713	0.0373	3	7.9	170.4	19.72	3	35*
0.74	1.192	0.0156	3	36*	34.7	1.65	3	87*
2.8	0.483	0.0993	3	74*	3.4	1.33	3	99*
9.9	0.127	0.2202	3	93*	0.7	1.24	3	100*

* - effect was statistically significant

Validity criteria fulfilled:

yes

Conclusions:

The test item 72h-ErC50 for growth rate reduction was 1.2 mg/L (C.I. 1.0 – 1.4 mg/L) based on TWA concentrations. The corresponding ErC10 was 0.21 mg/L (C.I. 0.15 – 0.27 mg/L) and the NOEC was 0.16 mg/L.

Executive summary:

The algal growth inhibition to Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a combined limit/range finding study at 1.0, 10.0 and 100.0 mg/L nominal concentrations, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed with a concentration of 0 (control), 0.10, 0.32, 1.0, 3.2 and 10 mg/L. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Environmental conditions were within acceptable limits. The concentrations of the test item were analytically verified via HPLC-DAD at 0 hours (test start), 24 hours and at 72 hours (test end) of the exposure. The measured concentrations at the start of test were 0.081, 0.29, 0.93, 3.0 and 9.4 mg/L in solutions prepared at 0.10, 0.32, 1.0, 3.2 and 10 mg/L, respectively. During the 72-hour exposure period, the measured concentration in the test solution prepared at 0.10 mg/L had decreased below the limit of detection (LOD; 0.029 mg/L). The remaining measured concentrations were at 19-106% relative to initial. Based on these results, effect parameters were based on Time Weighted Average (TWA) concentrations calculated to be 0.050, 0.16, 0.74, 2.8 and 9.9 mg/L. A dose-related increase of growth rate and yield inhibition was recorded at TWA concentrations of 0.16 mg/L and higher at the end of the test. The study met the acceptability criteria and was considered valid. The test item 72h-ErC50 for growth rate reduction was 1.2 mg/L (C.I. 1.0 – 1.4 mg/L) based on TWA concentrations. The corresponding ErC10 was 0.21 mg/L (C.I. 0.15 – 0.27 mg/L) and the NOEC was 0.16 mg/L.

Description of key information

EC50 (aquatic algae; growth rate) = 1.2 mg/L (C.I. 1.0 – 1.4) based on TWA concentrations, 72 hour, freshwater, OECD TG 201, 2018

EC10 (aquatic algae; growth rate) = 0.21 mg/L (C.I. 0.15 – 0.27) based on TWA concentrations, 72 hour, freshwater, OECD TG 201, 2018

NOEC (aquatic algae; growth rate) = 0.16 mg/L based on TWA concentrations, 72 hour, freshwater, OECD TG 201, OECD TG 201, 2018

Key value for chemical safety assessment

EC50 for freshwater algae:

1.2 mg/L

EC10 or NOEC for freshwater algae:

0.16 mg/L

Additional information

Key study: OECD TG 201, 2018 : The algal growth inhibition to Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. Following a combined limit/range finding study at 1.0, 10.0 and 100.0 mg/L nominal concentrations, a definitive study was conducted under static conditions with an initial cell density of 1.0x10^4 cells/mL. Five test item solutions were employed with a concentration of 0 (control), 0.10, 0.32, 1.0, 3.2 and 10 mg/L. Three replicates were tested for each test item concentration and six replicates for the control under constant illumination and shaking at a temperature of 22 ± 1 °C. Environmental conditions were within acceptable limits. The concentrations of the test item were analytically verified via HPLC-DAD at 0 hours (test start), 24 hours and at 72 hours (test end) of the exposure. The measured concentrations at the start of test were 0.081, 0.29, 0.93, 3.0 and 9.4 mg/L in solutions prepared at 0.10, 0.32, 1.0, 3.2 and 10 mg/L, respectively. During the 72-hour exposure period, the measured concentration in the test solution prepared at 0.10 mg/L had decreased below the limit of detection (LOD; 0.029 mg/L). The remaining measured concentrations were at 19-106% relative to initial. Based on these results, effect parameters were based on Time Weighted Average (TWA) concentrations calculated to be 0.050, 0.16, 0.74, 2.8 and 9.9 mg/L. A dose-related increase of growth rate and yield inhibition was recorded at TWA concentrations of 0.16 mg/L and higher at the end of the test. The study met the acceptability criteria and was considered valid. The test item 72h-ErC50 for growth rate reduction was 1.2 mg/L (C.I. 1.0 – 1.4 mg/L) based on TWA concentrations. The corresponding ErC10 was 0.21 mg/L (C.I. 0.15 – 0.27 mg/L) and the NOEC was 0.16 mg/L.