5-oxo-DL-proline

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2012 to 27 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Reason / purpose for cross-reference:

other: read-across target

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

Qualifier:

according to guideline

Guideline:

other: JMAFF in Japan (12-Nousan-No.8147, 2-7-7, 24 November, 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations and the control were sampled for analysis.
- Sampling method: Samples were collected at the beginning of the test and then at 24 hour intervals. In all cases, determination of test material concentration was only performed on one replicate vessel at each test concentration and from the additional sample prepared without algae. The sample without algae was performed in order to distinguish degradation and adsorption of the test material to algae.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was prepared at 100 mg/L by dilutioing 200 mg of the test material in 2000 mL 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, see below:

Nominal Concentration (mg/L) Amount of Stock Solution (mL) Amount of OECD Medium (mL)
100 1000 -
50 350 q.s ad 700
25 175 q.s ad 700
12.5 87.5 q.s ad 700
6.25 43.75 q.s ad 700
*q.s ad = quantum sufficiat ad (at sufficient quantity to make)

- Controls: Algal growth medium was inoculated with algal cells (without test material) and was examined in parallel to the test material concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG
- Pre-culturing: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 10^7 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature was in the range of 22.7 – 23.2 °C measured in the flask and between 22.5 and 23.9 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the study, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.71 – 9.20 during the experiment.

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
Measured concentrations in the presence of algae: 0.6, 0.9, 1.3, 1.8 and 2.6 mg/L
Measured concentrations in the absence of algae: 6.5, 13.4, 26.8, 54.0 and 108.2 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks, covered with air-permeable stoppers.
- Fill volume: 100 mL algal suspension per replicate.
- Shaking: Flasks were continuously shaken to keep algae in suspension.
- Initial cells density: 10^4 cells/mL
- Control end cells density: Mean cell number at 72 hours 70.50 ± 2.1 x 10^4 cells/mL.
- No. of vessels per concentration: Six replicates per test concentration.
- No. of vessels per control: Six replicates.
- Replicate uses: Three replicates were used for the observations (effect parameters) and two replicates for the 24 and 48 hour analytical measurements, the additional sample was used for further analytical measurements (at 0, 24, 48 and 2 hours) without algae in order to distinguish degradation and adsorption of the test material to algae.

GROWTH MEDIUM
- Standard medium used: Yes, OECD medium.

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: The algal culture flasks were continuously illuminated.
- Light intensity and quality: Approximately 8114 lux, provided by a 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: Determined at 24, 48 and 72 hours by manual cell counting using a microscopic method with a counting chamber.
- Microscopic observations: Performed at 24, 48 and 72 hours to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: A separation factor of 2.0 was employed for the definitive test concentration range.
- Range finding study: A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test material plus a control, for 72 hours. The test was performed with two replicates per test concentration and three replicates in the control group.
During the formulation procedure the test solutions were prepared by similar method as it was used in the main test.
- Test concentrations: Untreated control (0.0), 0.1, 1.0, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: The average cell number at 72 hours for exposure concentrations of 0.0, 0.1, 1.0, 10.0 and 100.0 mg/L were as follows: 73.67, 71.00, 71.50, 70.50 and 26.50 x 10^4 cell/mL, respectively.
Based on the significant toxic response observed during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.0 and one control were used in the main test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

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: Calculated as 282.92 mg/L (95 % confidence limits 150.19 - 532.96 mg/L)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

68.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence limits 57.65 – 82.26 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

86.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence limits 68.43 – 109.78 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Results:
> Growth Rate
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 in the concentration range of 50 to 100 mg/L, see Tables 2 and 3.
> Areas under the Growth Curve
The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value in the concentration range of 25 to 100 mg/L (nominal), see table 4.
> Yield
The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value in the concentration range of 25 to 100 mg/L (nominal), see Table 5.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The 72h ErC 50 : 0.98 mg/L, (95 % confidence limits: 0.91 – 1.05 mg/L)
The 72h EbC 50 : 0.70 mg/L, (95 % confidence limits: 0.65 – 0.76 mg/L)
The 72h EyC 50 : 0.68 mg/L, (95 % confidence limits: 0.62 – 0.73 mg/L)

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using a spreadsheet.
The ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis using statistical sofatware.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) using the same statistical software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Analytical Measurements

The test material was measured in samples containing algae at the start of experiment and after 24 hours and concentrations were found very close to the nominal. After 48 and 72 hours (at the end of the experiment) the test material could not be detected (i.e. was below the limit of detection 0.06 mg/L), therefore the corresponding measured geometric mean test material concentrations were: 0.6, 0.9, 1.3, 1.8 and 2.6 mg/L in presence of algae and 6.5, 13.4, 26.8, 54.0 and 108.2 mg/L in absence of algae, see Table 1.

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition.

Based on these results, the test material was considered to have absorbed to the increasing algal biomass, and should not be considered as lost from the test system.

Therefore it is recommended in the guideline to express the EC50 and NOEC/LOEC values in nominal concentrations.

Table 1: Measured Concentrations with 95 % Confidence Intervals in the Absence and Presence of Algae

Sample Concentration (mg/L)	Measured Concentrations (mg/L) In the Absence of Algae				Measured Concentrations (mg/L) In the Presence of Algae
	Start	Day 1	Day 2	End	Start	Day 1	Day 2	End
Control	ND	-	-	-	ND	-	-	-
6.25	6.7 ± 0.41	6.8 ± 0.19	6.5 ± 0.04	6.1 ± 0.04	6.7 ± 0.41	6.4 ± 0.05	ND	ND
12.5	13.5 ± 0.89	13.8 ± 0.26	12.9 ± 0.15	13.4 ± 0.56	15.5 ± 0.89	13.2 ± 0.44	0.06*	0.06*
25	27.6 ± 1.93	26.9 ± 0.68	26.4 ± 1.04	26.3 ± 0.46	27.6 ± 1.93	26.4 ± 0.64	0.06*	0.06*
50	54.0 ± 1.62	55.0 ± 1.60	53.0 ± 2.00	54.0 ± 2.67	54.0 ± 1.62	54.0 ± 1.03	0.06*	0.06*
100	108.0 ± 3.04	108.0 ± 7.18	109.0 ± 3.01	108.0 ± 4.71	108.0 ± 3.04	110.0 ± 1.09	0.06*	0.06*

ND = Not Detected, * concentration below the Limit of Detection.

Table 2: Average Specific Growth Rates (section-by-section) During the Test Period

Nominal Concentration (mg/L)	Average Specific Growth Rate for Three Replicates (d^-1)
	Day 0-1	Day 1-2	Day 2-3
6.25	1.2904	1.6865	1.2423
12.5	1.2904	1.6139	1.3146
25	1.2904	1.4607	1.4231
50	1.1945	1.5323	0.8891
100	1.0986	1.4130	0.7955

Table 3: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Nominal Concentration (mg/L)	Growth Rate (µ) and % Inhibition of µ
	0-24 hours		0-48 hours		0-72 hours
	µ	%	µ	%	µ	%
Control	0.0601	0.0	0.0616	0.0	0.0591	0.0
6.25	0.0538	10.6	0.0620	-0.7	0.0586	0.8
12.5	0.0538	10.6	0.0605	1.8	0.0586	0.9
25	0.0538	10.6	0.0573*	7.0	0.0580	1.9
50	0.0498	17.2	0.0568*	7.8	0.0502*	15.0
100	0.0458	23.9	0.0523*	15.1	0.0459*	22.3

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

Table 4: Area under the Growth Curves (A) and Percentage Inhibition of A

Nominal Concentration (mg/L)	Concentration
	0-24 hours		0-48 hours		0-72 hours
	A	%	A	%	A	%
Control	40.0	0.0	300.0	0.0	1354.0	0.0
6.25	32.0	20.0	288.0	4.0	1316.0	2.8
12.5	32.0	20.0	272.0	9.3	1284.0	5.2
25	32.0	20.0	240.0	20.0	1184.0*	12.6
50	28.0	30.0	228.0*	24.0	836.0*	38.3
100	24.0	40.0	184.0*	38.7	636.0*	53.0

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

Table 5: Yield (Y) and Percentage Inhibition of Y

Nominal Concentration (mg/L)	Concentration
	0-72 hours
	Y	%
Control	69.5	0.0
6.25	67.0	3.6
12.5	67.0	3.6
25	64.0*	7.9
50	36.3*	47.7
100	26.3*	62.1

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

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, significant inhibition of algal growth in comparison to the control was observed at concentrations ≥ 50 mg/L. Therefore the NOEC was determined as 25 mg/L and LOEC was determined as 50 mg/L. At the highest concentration tested 22.3 % growth rate inhibition was observed; therefore the EC50 was considered to be greater than 100 mg/L.
The 0-72 h areas and yield were significantly different from that of the control group in the examined concentration range of 25 - 100 mg/L (nominal), therefore the NOEC was determined as 12.5 mg/L and LOEC was determined as 25 mg/L (nominal).

Executive summary:

The toxicity of the test material to algae was determined in a growth inhibition study performed under GLP conditions and in line with the standardised guidelines OCED 201, EU Method C.3 and EPA OPPTS 850.5400.

Pseudokirchnerella subcapitata were exposed to the test material at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L for 72 hours under freshwater static conditions. Analytical verification of the test concentrations showed that the test material absorbed to the increasing algal biomass. Therefore effect concentrations have been reported in terms of nominal concentrations.

Under the conditions of the test, significant inhibition of algal growth was observed at concentrations ≥ 50 mg/L. Therefore the NOEC was determined as 25 mg/L and LOEC was determined as 50 mg/L. At the highest concentration tested 22.3% growth rate inhibition was observed, the EC50 was therefore considered to be greater than 100 mg/L.

The 0-72 h areas and yield were significantly different from that of the control group in the examined concentration range of 25 - 100 mg/L (nominal), therefore the NOEC was determined as 12.5 mg/L and LOEC was determined as 25 mg/L (nominal).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across from a structurally similar substance. Please refer to the RAAF report (section 13) for further information/ justification.

Reason / purpose for cross-reference:

read-across source

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: Calculated as 282.92 mg/L (95 % confidence limits 150.19 - 532.96 mg/L)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

68.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95 % confidence limits 57.65 – 82.26 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72

Dose descriptor:

EC50

Effect conc.:

88.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence limits 68.43 – 109.78 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Description of key information

Read-across to a substance with a similar structure.

Growth rate: 72 hour ErC50 > 100mg/L, NOEC 25 mg/L, LOEC 50 mg/L; Yield: 72 hour NOEC 12.5 mg/L, LOEC 25 mg/L, OECD 201, EU Method C.3, EPA OPPTS 850.5400, Sipos (2013b).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L

Additional information

Read-across to a substance with a similar structure.

In the key study (Sipos, 2013b) the toxicity of the read-across material to algae was determined in a growth inhibition study performed under GLP conditions and in line with standardised guidelines. The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as defined in Klimisch (1997).

Pseudokirchnerella subcapitata were exposed to the test material at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L for 72 hours under freshwater static conditions. Analytical verification of the test concentrations showed that the test material absorbed to the increasing algal biomass. Therefore effect concentrations have been reported in terms of nominal concentrations.

Under the conditions of the test, significant inhibition of algal growth was observed at concentrations ≥ 50 mg/L. Therefore the NOEC was determined as 25 mg/L and LOEC was determined as 50 mg/L. At the highest concentration tested 22.3 % growth rate inhibition was observed, the ErC50 was therefore considered to be greater than 100 mg/L.

The 0-72 h areas and yield were significantly different from that of the control group in the examined concentration range of 25 - 100 mg/L (nominal), therefore the NOEC was determined as 12.5 mg/L and LOEC was determined as 25 mg/L (nominal).