7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

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:

11-14 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 201. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Water solubility: 13.7 g/L at 20 °C [Experimental data (Safepharm, 2008), OECD 105 (flask method)]
- Vapour pressure: 0.178 Pa at 25 °C [Experimental data (Harlan, 2009), analogue data, OECD 104 (gas saturation method)]
- Log Kow: 1.95 at 30 °C [Experimental data (Safepharm, 2008), OECD 117 (HPLC method)]
- Relative density: 1.35 at 20°C [Experimental data (Safepharm, 2008), OECD 109 (pycnometer method)]
- Stability in water: Hydrolytically stable at environmentally relevant pHs (internal experimental Sponsor data)

Analytical monitoring:

yes

Details on sampling:

Analytical measurement was performed at the applied test concentration levels and from the control at the beginning and at the end of the experiment.
The samples were analysed by HPLC system.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal concentration of 100 mg/L was prepared with the test item and algal growth medium (OECD Medium). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae
- Controls: Algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
- Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

The temperature was 23.0 °C measured in the flask and between 22.9 and 23.4 °C measured within the climate chamber.

pH:

7.63 – 8.98

Nominal and measured concentrations:

Nominal concentrations: 9.53, 17.15, 30.86, 55.56 and 100 mg/L
Measured concentrations: 8.27, 14.75, 27.20, 49.65 and 91.88 mg/L. See table 6.1.5/1 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: closed; flasks were covered with air-permeable stoppers
- Aeration: Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- Initial cells density: 10000 algal cells per mL test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

OTHER TEST CONDITIONS
- Photoperiod: Algal culture flasks were continuously illuminated
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8145 lux (equivalent to ~110 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24, 48 and 72 h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- At the request of the Sponsor a range-finding test was not performed and five test concentrations in a geometric series with a separation factor of 1.8 and one untreated control were tested in the main experiment.
The following nominal concentrations were tested: 9.53, 17.15, 30.86, 55.56 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

83.54 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 67.25 – 103.77 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: analytically confirmed nominal

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

55.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

See table 6.1.5/2 in "Any other information on results incl. tables".

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 15/428-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 27 – 30 October 2015.
The 72 h ErC 50: 0.84 mg/L, (95 % confidence limits: 0.77 – 0.92 mg/L)
This value is within the range of laboratory ring test data.

Table 6.1.5/1: Geometric mean of the measured concentrations

Nominal concentrations, mg/L	Measured concentrations at the start (t0h), mg/L	Measured concentrations at the end (t72h), mg/L	Geometric mean measured concentrations, mg/L	Percentage of the nominal concentrations
Control	-	-	-	-
9.53	9.95	6.87	8.27	86.7
17.15	17.70	12.29	14.75	86.0
30.86	32.17	23.00	27.20	88.1
55.56	58.56	42.10	49.65	89.4
100	105.6	79.95	91.88	91.9

AVERAGE SPECIFIC GROWTH RATES

The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the two highest concentrations of 55.56 and 100 mg/L (nominal), however the observed effect (inhibition: μ = 3.8 %) at 55.56 mg/L is not biologically significant, but is due to the biological variability of the test system. Therefore the No Observed Effect Concentration (NOEC) was determined as 100 mg/L (nominal).

The 72 h E_rC₁₀ value was calculated [by Probit analysis (TOXSTAT software)] as 83.54 mg/L (95 % confidence limits: 67.25 – 103.77 mg/L) (based on analytically confirmed nominal concentrations).

The 72 h E_rC₅₀ value could not be calculated, it was determined as higher than the highest tested concentration level of 100 mg/L (nominal).

 

Table 6.1.5/2: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

 

Concentration			Growth rate (μ) and % inhibition ofμ
Nominal [mg/L]	Measured [mg/L]	% of nominal	0–24 h		0–48 h		0–72 h
			μ	%	μ	%	μ	%
Control	0.0	-	0.0593	0.0	0.0607	0.0	0.0597	0.0
9.53	8.27	86.7	0.0609	-2.6	0.0609	-0.3	0.0599	-0.3
17.15	14.75	86.0	0.0609	-2.6	0.0606#	0.3	0.0597	0.1
30.86	27.20	88.1	0.0609	-2.6	0.0594	2.2	0.0593	0.8
55.56	49.65	89.4	0.0578	-2.6	0.0598	1.5	0.0575*	3.8
100.0	91.88	91.9	0.0538	9.3	0.0539*	11.3	0.0512*	14.3

 

* : statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

#: at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

 

VALIDITY

The cell density in the control cultures increased by the factor of 73.83 within three days.

The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 6.10 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.94 %.

All validity criteria were met, therefore the study can be considered as valid.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 72h-ErC10 and the 72h-ErC50 values were determined to be 83.54 mg/L (95% CL 67.25-103.77 mg/L) and > 100 mg/L, respectively, based on analytically confirmed nominal concentrations.

Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item over an exposure period of 72 hours.

At the request of the Sponsor a range-finding test was not performed and five test concentrations in a geometric series with a separation factor of 1.8 and one untreated control were tested in the main experiment. The nominal concentrations of test item used in the main experiment were: 9.53, 17.15, 30.86, 55.56 and 100.0 mg/L. The corresponding measured geometric mean test item concentrations, analytically determined at the start and the end of the experiment, were: 8.27; 14.75; 27.20; 49.65 and 91.88 mg/L.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

At the request of the Sponsor, as the geometric mean measured concentration deviated not more than 20 per cent from the nominal concentrations, the biological results are based on the analytically confirmed nominal concentrations.

Under the test conditions, the 72h-ErC10 and the 72h-ErC50 values were determined to be 83.54 mg/L (95% CL 67.25-103.77 mg/L) and > 100 mg/L, respectively, based on analytically confirmed nominal concentrations.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokirchneriella subcapitata) > 100 mg/L (analytically confirmed nominal concentration)

72h-ErC10 (Pseudokirchneriella subcapitata) = 83.54 mg/L (95% CL: 67.25 -103.77 mg/L; analytically confirmed nominal concentration)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

83.54 mg/L

Additional information

One key study is available to assess the influence of the registered substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata in a 72h static condition, according to the OECD Guideline 201 and in compliance with GLP. The nominal concentrations of test substance used in the main experiment were: 9.53, 17.15, 30.86, 55.56 and 100.0 mg/L. The corresponding measured geometric mean test item concentrations, analytically determined at the start and the end of the experiment, were: 8.27; 14.75; 27.20; 49.65 and 91.88 mg/L. As the geometric mean measured concentration deviated not more than 20 per cent from the nominal concentrations, the biological results are based on the analytically confirmed nominal concentrations. Under the test conditions, the 72h-ErC10 and the 72h-ErC50 values were determined to be 83.54 mg/L (95% CL 67.25-103.77 mg/L) and > 100 mg/L, respectively, based on analytically confirmed nominal concentrations.