L-Glutamic acid, N-(15-carboxy-1-oxopentadecyl)-, 5-(2,5-dioxo-1-pyrrolidinyl) ester

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:

2009.12.01 to 2009.12.17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed in accordance with OECD principles of GLP with no deviations. Furthermore, the study fulfilled the validity criteria described in the OECD guideline.

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2011

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

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

PC2414

IUPAC Name:

PC2414

Details on test material:

- Name of test material (as cited in study report): PC2414
- Lot/batch No.: 0932L004
- Expiration date of the lot/batch: 2011-03-0

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
NA

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

PC2414 was tested in the following nominal concentrations: 0, 10, 20; 50; 100; 200 and 400 mg/L.. Exposure concentrations of PC2414 should be maximised by keeping duration of the media preparation stage to a minimum according to the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures. However, PC2414 was expected to hydrolyse rapidly and therefore, the test was mainly testing the toxicity of the hydrolysis products (NHS and impurity No.4).

In order to verify the tested concentrations, duplicate samples of each test concentration were taken at the initiation of the test, every 24 hours and at the termination of the test. Samples of approx. 40 mL were collected in 50-mL glass bottles and stored at -20 ± 2°C. Both samples with algae and without algae were taken. Samples from all test concentrations were analysed.

Test solutions

Vehicle:

no

Details on test solutions:

A stock solution of 455 mg/L was prepared by weighing out 910 mg PC2414 and dissolving it in 2,000 mL of algae test medium. The stock solution was prepared with continuous pH adjustment during 6 hours. pH was adjusted to 8.0 by adding 32 droplets of 6 M NaOH. The test solution was stirred for 6 hours by means of a magnet stirrer. The test solutions were prepared by diluting the stock solution in the algae test medium. In the dilution series a stable pH of 7.9 was then achieved indicating that PC2414 was hydrolysed.

Freshly produced ISO-medium was used as growth medium in the test. The medium was prepared from Milli-Q water and salts were added to the water according to standard procedure.

A test with the reference substance potassium dichromate was performed in order to verify the sensitivity of the algae. The reference test was performed at the following concentrations: 0 (control); 0.10; 0.20; 0.40; 0.70; 1.0; 1.4 and 2.0 mg/L.

Test organisms

Test organisms (species):

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

Details on test organisms:

The toxicity of PC2414 was determined as the growth inhibition of the freshwater alga Pseudokirchneriella subcapitata (clone: NIVA, CHL 1). The algae Pseudokirchneriella subcapitata is cultured at DHI under the test conditions prior to the start of the test. Algae from this culture were used in the study.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

NA

Test conditions

Hardness:

250 ± 25 mg/L expressed as CaCO3

Test temperature:

23.2 ± 0.2°C

pH:

Control group: 7.9 (start), 9.3 (end)
Higest concentration group: 8.1 (start), 7.6 (end)

Dissolved oxygen:

NA

Salinity:

NA

Nominal and measured concentrations:

The results of the chemical analysis of the main hydrolysis product (Imp. No. 4) of PC2414 showed an average recovery after 72 hours of 0-37% of the analysed concentration of the hydrolysis product at the beginning of the test (Table 1).

Details on test conditions:

The test was performed in glass flasks with wide neck and a total capacity of 250 mL, each containing 100 mL of test solution consisting of algal growth medium, algae and test product. The design included triplicate test flasks of each concentration, six controls (flasks with algae but no test product) and two blanks of each concentration (flasks with test product but no algae).Furthermore, three flasks of the control and three flasks of each test concentration without algae were used to collect samples for chemical analysis. One flask of the control and each test concentration were harvested at each sampling time (approx. 24h; 48h and 72h).
 
The algae were incubated for approx. 72 hours under continuous shaking (approx. 120 RPM) at 23.2 ± 0.2°C and constant illumination from a panel of fluorescent light with an intensity of approx. 60-120 µmol*m-2*sec-1.
 
The cell density was approx. 9,600 cells/mL at test start, measured in the control. The algae growth was followed as in-vivo fluorescence and as number of particles at the beginning of the test and after 24, 48 and 72 ± 2 hours of incubation in triplicate test flasks, the blanks and the six controls. The fluorescence was measured by use of a Turner TD-700 Laboratory Fluorometer and the number of particles by use of a Beckman Coulter MultisizerTM3 Coulter Counter®.
 
The identity and normal appearance of Pseudokirchneriella subcapitata in the control was confirmed at the end of the test by microscopy.

Reference substance (positive control):

yes

Remarks:

Analytical grade potassium dichromate (Sigma-Aldrich 31255, CAS No. 7778-50-9, Lot No. 62420)

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

No linear dose-response relationship was found in the test. Due to the lack of a linear dose-response relationship, EC values for PC2414 could not be calculated. However, the EC50 value can be estimated to be > 200 mg/l. A No Observed Effect Concentration (NOEC) of <10 mg/L can be derived from the test data.

Results with reference substance (positive control):

Inhibitory effect on the algal growth was observed after 72 hours, thereby validating the study design.The EC50 of the positive control potassium dichromate (K2Cr2O7) was 1.02 mg/L (95% confidence interval: 0.98- 1.06) was not significantly different from the results obtained n an international ring test in 1981 (EC50 = 1.19 (0.65-1.73) mg/L), and the NOEC 0.2 mg/L.

Reported statistics and error estimates:

The growth inhibition was determined on the basis of the growth rates expressed as fluorescence or particle counts measured in the individual test flasks compared to the control.

The concentrations of potassium dichromate inhibiting the growth rate 10% and 50% were expressed as EC10 and EC50 according to the ISO standard. The EC values were calculated by use of the computer program ‘TOXEDO’.

Any other information on results incl. tables

Table 1. Analytical test results

Nominal concentration (mg PC2414/L)	Sampling time (hours)	Imp no. 4 (mg/L)	Imp. no. 4 in % of initial conc. (%)
10	0	7.4	100
10	24	7.0	94
10	48	2.0	27
10	72	0	0
20	0	14.3	100
20	24	14.0	98
20	48	4.2	30
20	72	0	0
50	0	35.1	100
50	24	36.3	103
50	48	12.6	36
50	72	0	0
100	0	69.1	100
100	24	68.7	99
100	48	24.7	36
100	72	12.2	18
200	0	145.1	100
200	24	101.1	70
200	48	61.1	42
200	72	54.2	37
400	0	372.0	100
400	24	403.4	108
400	48	110.1	30
400	72	125.1	34

The concentration of Imp. No. 4 was somewhat stable in all test concentrations except 200 mg/L in the first 24 hours of the test period. Between 24 hours and 48 hours, a clear decline in the concentration was observed and at 72 hours only 0-37% of the initial concentration was present in the test solutions. The concentration of Imp. No. 4 was measured both in samples with algae and in samples without algae. The analytical results show that the concentration in the samples with algae is lower than in samples without algae indicating that the algae have an influence on the analytical test results.

No linear dose-response relationship was observed. However, the EC50 value can be estimated to be > 200 mg/L.

 

Exposure of Pseudokirchneriella subcapitata to PC2414 resulted in some growth inhibition that was significantly different to the control group.No linear dose-response relationship was observed. However, the 72 hour E_rC50 and E_bC50 toxicity values could be estimated to be > 200 mg/L. Based on visual inspections the colour of the algae in the highest tested concentration seemed to be affected (less green) but this was not reflected in the fluorescence measurements. However, the particle counts showed a different pattern, with a significant difference between the control and the highest tested concentration but still the non-linear dose-response relationship is not allowing statistical calculations of effect concentrations.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

PC2414 was shown to inhibit the growth rate of the micro alga Pseudokirchneriella subcapitata at lowest tested concentration, hence a No Observed Effect Concentration (NOEC) of <10 mg/L could be derived. The 72 hour ErC50 and EbC50 toxicity values could be estimated to be > 200 mg/L

Executive summary:

PC2414 was tested for inhibitory effect on the growth of the micro alga Pseudokirchneriella subcapitata according to OECD Guideline 201 and EUMethodC.3 which is further specified in the ISO International Standard 8692. The test was carried out in compliance with the OECD Principles of Good Laboratory Practice (GLP). PC2414 was tested at the following nominal concentrations: 0 (control); 10; 20; 50; 100; 200 and 400 mg/L. PC2414 are rapidly hydrolysed when mixed in water and therefore, the test was mainly testing the toxicity of the hydrolysis products (NHS and Impurity No. 4).

No linear dose-response relationship was found in the test. However, the 72 hour E_rC50 and E_bC50 toxicity values could be estimated to be > 200 mg/L.

All validity criteria were achieved. Therefore, the conditions of the test were considered acceptable. The non-linear dose-relationship is most probable caused by the change of the test product from PC2414 into hydrolysis products, presuming that PC2414 and its hydrolysis products are having different toxicity to the algae.

A No Observed Effect Concentration (NOEC) of < 10 mg/L could be derived.