Pentanedioic acid, 2-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-, (2S)-

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:

22 June 2015 - 25 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-(1,3-DIOXO-2,3-DIHYDRO-1H-ISOINDOL-2-YL)PENTANEDIOIC ACID

EC Number:

608-945-6

Cas Number:

340-90-9

Molecular formula:

C13H11NO6

IUPAC Name:

(2S)-2-(1,3-DIOXO-2,3-DIHYDRO-1H-ISOINDOL-2-YL)PENTANEDIOIC ACID

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Identification : Phthalyl-L-Glu
- Appearance: White powder
- Batch: 10844200 (taken from label)
- Purity/Composition: 99.2 weight-% (average content)
- Test substance storage: At room temperature
- Stable under storage conditions until: Not indicated

- Purity/composition correction factor: No correction factor required
- Test substance handling: No specific handling conditions required
- CAS Number: 340-90-9
- Volatile: Not indicated
- Solubility in water: Not available
- Stability in water: Not available

Sampling and analysis

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency: at t=0 h, t=24 h and t=72 h
Volume: 4.0 ml
Storage: Samples were stored in a freezer until analysis.

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

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate 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.

Additionally, reserve samples of 4.0 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with a the highest concentration of 100 mg/l applying 23 minutes of magnetic stirring to accelerate the dissolving of the test substance in the test medium and ensure homogeneity. The pH of this solution was 5.3 and adjusted to 6.1 using 1M NaOH (Merck, Darmstadt, Germany) The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

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 10E4 cells/ml.

Test organisms

Test organisms (species):

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

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation):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 10E4 cells/ml.
- 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 (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C, except for a short period in which the temperature was 20°C

pH:

pH between 6.7 and 8.2 (control and 100 mg/l).

Nominal and measured concentrations:

- Nominal concentration: 0.10, 1.0, 10 and 100 mg/l

- Measured concentration:
The samples from 100 mg/l were analyzed. The actual concentrations at the start was 96 mg/l and remained stable and within nominal during the test (89% of initial, 85% of nominal). Consequently the parameters were reported in terms of the analytical confirmed nominal concentration.

Details on test conditions:

COMBINED LIMIT/RANGE-FINDING TEST:

Since, no effects on growth rate were observed at the highest concentration the test was used as a limit test.

TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1780000 cells/ml

Replicates:
- 6 replicates of the control,
- 6 replicates of 100 mg/l,
- 3 replicates of each lower concentration,
- 1 or 2 replicates of each concentration without algae,
- 1 extra replicate of each concentration for sampling purposes.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 79 to 82 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h ErC10,50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: control and 0.1, 1.0, 10 and 100 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: analytical confirmed nominal concentration

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: analytical confirmed nominal concentration

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: analytical confirmed nominal concentration

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: analytical confirmed nominal concentration

Details on results:

- Exponential growth in the control :yes
- Observation of abnormalities: no abnormalities observed
- Unusual cell shape: no
- Colour differences: no

Results with reference substance (positive control):

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.3 mg/l with a 95% confidence interval ranging from 1.2 to 1.5 mg/l.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.46 mg/l with a 95% confidence interval ranging from 0.43 to 0.49 mg/l.

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 yield (STUDENT-t test for Homogeneous Variances, a=0.05, one-sided, smaller).

For growth rate statistical analysis of the data was not needed as the effects recorded biologically not significant (<10%).

The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Phthalyl-L-Glu inhibited the yield of this fresh water algae species significantly at 100 mg/l, no inhibition of growth rate was recorded at any of the concentrations of Phthalyl-L-Glu tested.

The EC50 for growth rate (72h-ERC50) and yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/l.

The 72h-NOEC for growth rate inhibition was 100 mg/l.