2-[(3S)-2,6-dioxooxan-3-yl]isoindole-1,3-dione

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 August 2016 - 26 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23.

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[(3S)-2,6-dioxooxan-3-yl]isoindole-1,3-dione

EC Number:

607-813-5

Cas Number:

25830-77-7

Molecular formula:

C13 H9 N O5

IUPAC Name:

2-[(3S)-2,6-dioxooxan-3-yl]isoindole-1,3-dione

Test material form:

solid

Details on test material:

- Appearance: white solid
- Storage conditions: in refrigerator (2-8°C) protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0, t=24 and t=72
Volume: 3.2 mL
Storage: samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: A saturated solution (SS) was prepared at a loading rate of 100 mg/L applying three days of magnetic stirring to ensure maximum dissolution of the test item in test medium. The resulting aqueous mixture was filtered through a membrane and the obtained saturated solution was used as the highest test concentration. The pH was adjusted from 5.2 to 6.2 with a 1 M NaOH solution (Merck, Darmstadt, Germany). Hereafter, lower test concentrations were prepared by subsequent dilutions of the SS in test medium. Due to the light sensitivity of the test item, preparation of test solutions was performed under dimmed light and used glassware was wrapped in aluminum foil. All final test solutions were clear and colorless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

- Controls: test medium without test item or other additives

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture.

CULTIVATION:
- Method: 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/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Medium different from test medium: yes, M1

- Pre-culture: 3 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.
Pre-culture medium different from test medium: no, M2

ACCLIMATION
- Acclimation period: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3 mg/L

Test temperature:

21 - 23°C

pH:

5.3 - 8.0 throughout the test (see also table 1 in the section 'Any other information on materials and methods' for more details)

Nominal and measured concentrations:

Nominal test concentration: 1.0, 10 and 100 mg/L
Mean measured test concentration: 88 mg/L (only samples from the control and highest concentrations were analysed)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 165.6 x 10^4 cells/mL
- No. of vessels per control and highest test concentration (replicates): 6 + 1 for sampling after 24 hours
- No. of vessels per lower concentration (replicates): 3 + 1 for sampling after 24 hours
- 1 or 2 extra replicates of each concentration without algae were used

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1

OTHER TEST CONDITIONS
- Adjustment of pH: yes, pH was adjusted from 5.2 to 6.2 with a 1 M NaOH solution (Merck, Darmstadt, Germany)
- Light intensity and quality: continuous illumination using TLD-lamps with a light intensity within the range of 75 to 86 µE/m^2/s.

EFFECT PARAMETERS MEASURED: growth rate and yield after 72 hours.
- Additional measurements: pH at the beginning and at the end of the test. temperature of the medium continuously in a temperature control vessel, appearance of the cells at the end of the final test by microscopic observations.
- Determination of cell concentrations: At the beginning of the test, 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 with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions. Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (July 2016)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 88 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 88 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

88 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 88 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 88 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

< 88 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield

Details on results:

- Inhibition of growth rate at the highest concentration was statistically significant (p<0.05) but biologically not relevant ( < 10%), therefore NOEC was determined to be 88 mg/L. Inhibition of yield at the highest concentration was statistically significant (p<0.05) as well as biologically relevant and therefore the NOEC was determined to be < 88mg/L.
- Exponential growth in the control (for algal test): yes
- No biological, behavioural or other abnormalities were observed.
- Effect parameters were determined by the initially measured exposure concentrations because concentrations proved to remain stable during the 72 hours of exposure. Relative to initial loading rates ranged from 87 to 99%.

Temperature values remained within acceptable limits throughout the duration of the study. The pH was generally within the limits prescribed by the study plan except for the highest test concentration where the pH was 5.3. No observed significant effects on algal growth were reported.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.0 mg/L, 95%-CI: 0.97-1.0 mg/L
- EyC50: 0.43 mg/L, 95%-CI: 0.42-0.44 mg/L
- Other: results fell within the historical range.

Reported statistics and error estimates:

For the determination of the NOEC and the EC50, the approaches recommended in OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested).

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1         Concentrations of the test item in test medium

Time of sampling^1 [hours]	Percentage of SS^2 [%]	Analysed concentration [mg/L]	Relative to initial [%]
0	0	n.d.
	100	87.6
	100^3	88.4
24	0	n.d.	n.a.
	100	85.2	97
	100^3	87.4	99
72	0	0.034^4	n.a.
	100	84.2	96
	100^3	77.2	87

¹Samples were stored in the freezer (≤ -15°C) until the day of analysis.

² Percentage of a saturated solution prepared at a loading rate of 100 mg/L.

³Without algae.

⁴Estimated value, calculated by extrapolation of the calibration curve.

n.d.Not detected.

n.a.Not applicable

Table 2 Percentage inhibition of growth rate (total test period) during the test

Phthaloyl-L-glutamic acid anhydride % SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.702	0.0357	6
1.0	1.711	0.0432	3	-0.6
10	1.736	0.0519	3	-2.0
100 (88)	1.615	0.0223	6	5.1*^#

() - Between brackets, the measured concentration is given in mg/L.

* - effect was statistically significant

^#- effect biologically not relevant (<10%)

Table 3 Percentage inhibition of growth rate at different time intervals during the test

Phthaloyl-L-glutamic acid anhydride % SS prep. at 100 mg/L	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.063		1.559		1.483
100 (88)	6	1.949	5.5	1.617	-3.7	1.281	13.7

() - Between brackets, the measured concentration is given in mg/L.

Table 4 Percentage inhibition of yield during the test

Phthaloyl-L-glutamic acid anhydride % SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	164.6	18.28	6
1.0	169.5	21.71	3	-3.0
10	183.0	28.89	3	-11.1
100 (88)	126.5	8.78	6	23.2*

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1). In the control, cell density increased > 16 fold. 2). Mean CV for section-by-section specific growth rates in the control cultures < 35%. 3). CV of average specific growth rates during the whole test period in replicate control cultures < 7%.

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate was recorded at any of the concentrations of Phthaloyl-L-glutamic acid anhydride tested.
The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) were beyond the range tested, i.e. exceeded 88 mg/L, being the concentration measured in a filtrate prepared at a loading rate of 100 mg/L.
The 72h-NOEC for growth rate inhibition was at or above the limit of solubility (88 mg/L being the concentration measured in a filtrate prepared at a loading rate of 100 mg/L).
The 72h-NOEC for yield inhibition was found to be <88 mg/L, being the concentration measured in a filtrate prepared at a loading rate of 100 mg/L and considered the limit of solubility.

Executive summary:

 This study was performed to assess the effect of the test substance on the growth rate and yield of fresh water algae ( Pseudokirchneriella subcapitata ) after 72 hours of exposure. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 and GLP under static conditions.

Six exponentially growing algal cultures were exposed to an untreated control and the limit concentration, i.e. 100 mg/L. In addition, three replicates per group were exposed to 1.0 and 10 mg of test substance per litre in a combined limit/range-finding test. The initial algal cell density was 10⁴ cells/mL. The total exposure period was 72 hours. Samples from the control and the highest concentration were taken and analysed at the start, after 24 and 72 hours of exposure. Measured concentration at t=0 was 88 mg/L. Measured concentrations during the test ranged from 87 - 99 % of initial loading rates, therefore it was concluded that the test substance remained stable during the exposure period and the initial measured concentration was used to determine the effect parameters.

Only at the highest concentrations, an inhibition of growth rate and an inhibition of yield were observed. The inhibitions were statistically significant (p < 0.05). However, the inhibition of growth rate was biologically not relevant (< 10%). The NOEC for growth rate was therefore determined to be 88 mg/L. The NOEC for yield was determined to be < 88 mg/L. EC50 for inhibition of growth rate and inhibition of yield in algae after 72 hours of exposure could not be calculated because the test item proved to be non-toxic (EC50> maximum soluble concentration tested). EC50 for both growth rate and yield were determined to be > 88 mg/L.