Potassium (2S)-4-carboxy-2-(tetradecanoylamino)butanoate

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:

22 April 2013 to 25 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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: Japanese guidelines for studies on new chemical substance required by the Chemical Substance Control Law 1973, amended 2009 (YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002), partially amended 2006 (Japanese METI, MHLW and MOE)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-3, The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Analytical measurements were performed in the control and at all test concentrations.
- Sampling method: Treated samples were collected at the beginning of the test and then in 24 hour intervals throughout the experiment. Samples were collected from both flasks with and without algae. Control samples were collected at the start and end only.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of 200 mg test material was diluted in 2000 mL OECD medium (stock solution concentration: 100.0 mg/L). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae (see Table 'Preparation of Test Solution from Stock Solution').
- Controls: Untreated control, 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: Algae were sourced from a laboratory culture, whcih was cultures inder standard laboratory conditions.
- Method of cultivation: 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 10⁷ cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test temperature:

22.2 – 22.4 °C

pH:

7.01 (start pH for the nominal sample concentration of 100 mg/L) – 9.38
7.92 (start concentration for the control)

Dissolved oxygen:

NDA

Salinity:

N/A

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks.
- Type: Flasks were covered with air-permeable stoppers.
- Fill volume: 100 mL
- Aeration: The test medium was aerated prior to initiation.
- Initial cells density: 10⁴ cells/mL.
- Control end cells density: 68.17 ± 1.2 (x 10⁴ cells/mL)
- No. of vessels per concentration (replicates): Six replicates in total, three for observations, two for the 24 and 48 hour analytical measurements, and an additional sample without algae for further analytical measurements at 0, 24, 48 and 72 hours.
- No. of vessels per control (replicates): Six replicates in total (see above).

GROWTH MEDIUM
- Standard medium used: Yes, reconstituted algal growth medium (OECD medium, according to OECD 201).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium was used as dilution water.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Agitation: Flasks were continuously shaken.
- Photoperiod: Cultures were continuously illuminated.
- Light intensity and quality: Approximately 8209 lux, 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 %.

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.
- Other: Microscopic observation of the algal cells in each concentration and in the control was performed (at 24, 48 and 72 hours) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Test concentrations: 0.0 (untreated control), 0.1, 1.0, 10.0 and 100 mg/L.
- Results used to determine the conditions for the definitive study: A significant toxic response was observed in the preliminary study. The average number of cells (x 10⁴ cells/mL) at 72 hours in the preliminary test was 68.00, 69.00, 69.50, 70.50 and 1.00, at nominal concentrations of 0.0, 0.1, 1.0, 10.0 and 100.0 respectively.
Based on these results, five test concentrations in a geometric series with a separation factor of 2.0 and one control were selected for use in the main test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 25.37 - 30.40

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): Yes, mean cell numbers against time can be seen in the attachment in the 'Overall remarks, attachments' section.
- Unusual cell shape: Thin cells were observed at the concentration levels of 25, 50 and 100 mg/L (nominal), 72 hours after the start of the test.
- 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 in the concentration range of 12.5 and 100.0 mg/L (nominal) (see Table 'Growth Rates (µ) and Percentage Inhibition of µ during the Test Period' and Table 'Effect Parameters').
- Areas under the growth curves: 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 12.5 and 100.0 mg/L (nominal) (see Table 'Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period' and Table 'Effect Parameters').
- 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 12.5 and 100.0 mg/L (nominal) (see Table 'Yield (Y) and Percentage Inhibition of Y during the Test Period' and Table 'Effect Parameters').

Results with reference substance (positive control):

- Results with reference substance valid? The restuls were within the range of the laboratory and confirm the validity of the test.
- EC50:
> 72h ErC 50 : 0.87 mg/L, (95 % confidence limits: 0.79 – 0.96 mg/L)
> 72h EbC 50 : 0.48 mg/L, (95 % confidence limits: 0.44 – 0.53 mg/L)
> 72h EyC 50 : 0.45 mg/L, (95 % confidence limits: 0.41 – 0.50 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 computer software.
The ErC50, EbC50 and EyC50 values of the test amterial and their confidence limits were calculated using Probit analysis by statistical software (based on the calculated geometric mean concentrations).
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) by 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

Test material measurements taken in the presence of algae deviated more than 20 % from the nominal in most cases at 24 and 48 hours. After 72 hours the test material could only be detected at the highest nominal concentration level of 100 mg/L, at 50 mg/L the measured concentration was below the limit of quantification, and at all other nominal concentrations it could not be detected.

 

In order to calculate the mean exposure concentrations, where the measured concentration was below the Quantification Limit, the concentration was taken as the half of the Limit of Quantification (LOQ = 10.0 mg/L) and where the measured concentration was not detected, the end concentration was taken as the Limit of Detection (LOD = 2.0 mg/L) (according to OECD 23; paragraph 3.3). Therefore the corresponding measured geometric mean test material concentrations were: 4.2, 7.0, 11.6, 23.2 and 74.2 mg/L in presence of algae (see Table 'Analytical Measurements in the Presence and Absence of Algae').

 

The specific growth rate for tested concentrations of 6.25, 12.5 and 25 mg/L (nominal) was clearly reduced at the section days 2-3 and it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition (see Table 'Average Specific Growth Rates (Section-by-Section) During the Test Period').

 

In addition, results of the analytical measurements of samples in the absence of algae showed measured geometric mean test material concentrations of 6.6; 12.4; 24.0; 43.7 and 87.0 mg/L (see Table 'Analytical Measurements in the Presence and Absence of Algae') for the nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L respectively.

 

 

 

Based on these results most probably the test material is indeed 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.

 

Analytical Measurements in the Presence and Absence of Algae

Nominal Concentration (mg/L)	Measured Concentrations in the Presence of Algae (mg/L)				Geometric Mean (mg/L)	Measured Concentrations in the Absence of Algae (mg/L)				Geometric Mean (mg/L)
	Start	24 h	48 h	72 h		Start	24 h	48 h	72 h
Control	n.d.	-	-	n.d.	-	n.d.	-	-	n.d.	-
6.25	7.85	4.61	4.28	2.0**	4.2	7.85	6.31	5.96	6.58	6.6
12.5	14.3	8.37	9.87	2.0**	7.0	14.3	11.7	11.3	12.5	12.4
25.0	27.7	16.4	19.9	2.0**	11.6	27.7	21.3	21.9	25.5	24.0
50.0	51.1	30.4	37.2	5.0*	23.2	51.5	34.2	40.8	50.6	43.7
100.0	104	59.7	69.9	69.8	74.2	104	67.8	80.5	101	87.0

n.d. = not detected

* Concentration < LOQ. The concentration was taken as the half of the LOQ (10.0 mg/L)

** Concentration < LOD. The concentration was taken as the LOD (2.0 mg/L)

 

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	1.5351	1.4085	1.2661
12.5	1.2904	1.4809	1.0499
25	1.2904	1.0088	0.2260
50*	0.6931	0.0000	0.0000
100*	0.0000	0.0000	0.0000

* There were no increasing of the growth rates, because of the observed high effect

 

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	0.0640	0.0	0.0607	0.0	0.0586	0.0
6.25	0.0640	0.0	0.0613	-1.0	0.0585	0.3
12.5	0.0538	15.9	0.0577	4.9	0.0531*	9.5
25.0	0.0538	15.9	0.0479*	21.1	0.0351*	40.2
50.0	0.0289*	54.8	0.0144*	76.2	0.0096*	83.6
100.0	0.0000*	100.0	0.0000*	100.0	0.0000*	100.0

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

 

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Nominal Concentration (mg/L)	Concentration
	0 – 24 hours		0 – 48 hours		0 – 72 hours
	A	%	A	%	A	%
Control	44.0	0.0	298.0	0.0	1314.0	0.0
6.25	44.0	0.0	304.0	-2.0	1316.0	-0.2
12.5	32.0*	27.3	244.0*	18.1	960.0*	26.9
25.0	32.0*	27.3	172.0*	42.3	420.0*	68.0
50.0	12.0*	72.7	36.0*	87.9	60.0*	95.4
100.0	0.0*	100.0	0.0*	100.0	0.0*	100.0

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

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

Nominal Concentration (mg/L)	Concentration 0 - 72 hours
	Y	%
Control	67.2	0.0
6.25	66.3	1.2
12.5	44.7*	33.5
25.0	11.7*	82.6
50.0	1.0*	98.5
100.0	0.0*	100.0

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

 

pH

The pH values at the start and end of each of the tests (control, 6.25, 12.5, 25, 50 and 100 mg/L) were recorded and are given in Table 'pH-Values in the Test Media at the Start and End of the Test'. The pH of the control medium increased by a maximum of 1.46 units (7.92-9.38) during the study as presented in the table below. 

 

pH-Values in the Test Media at the Start and End of the Test

Concentration Nominal mg/L	pH Values
	Start	End
Control	7.92	9.35
		9.36
		9.35
		9.28
		9.38
		9.35
6.25	7.83	9.21
		9.28
		9.34
12.5	7.76	8.71
		8.78
		8.77
25.0	7.65	7.98
		8.02
		7.95
50.0	7.44	7.28
		7.34
		7.27
100.0	7.01	7.19
		7.25
		7.21

 

Effect Parameters

Parameter (0 – 72 hours)	Growth Rate (r) (mg/L)	Yield (y) (mg/L)	Biomass (b) (mg/L)
EC50	27.77	15.81	19.08
95% confidence limits	25.37 – 30.40	14.55 – 17.18	17.44 – 20.87
NOEC	6.25	6.25	6.25
LOEC	12.5	12.5	12.5

 

Validity criteria

The study fulfilled the validity criteria outlined in the most recent EU guidance (OECD 201, 2006:2011) as detailed below:

• Cell density in the controls should increase by at least a factor of 16 in 72-hours. In the test cell density increased by a factor of 68.17 after 72 hours.


• The coefficient of variation of sectional (daily) growth rates in the controls should be ≤ 35%. In the test this was 11.58%.


• The coefficient of variation of average growth between control replicates should be ≤ 7%. In the test this was 0.4%.

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on results incl. tables' for further information.

Conclusions:

With respect to the inhibitory effect of the test material, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 20.4 - 100.0 mg/L (nominal). Based on the growth rate of the algae the following effect parameters were determined, ErC50 27.77 mg/L, NOEC 6.25 mg/L, and LOEC 12.5 mg/L.

Executive summary:

The toxicity of the test material to algae was determined in a growth inhibition study performed with Pseudokirchneriella subcapitata under GLP conditions and in accordance with the standardised guidelines OECD 201, EU Method C.3 and EPA OPPTS 850. 5400.

Algal cultures were exposed to the test material at concentrations ranging from 6.25 to 100 mg/L for 72 hours under static freshwater conditions. Cell numbers were manually counted at 24, 48 and 72 hours. Microscopic observations of algal cells were also performed at 24, 48 and 72 hours to detect any abnormal appearance of the algae. The pH at the start for the nominal sample concentration of 100 mg/L was 7.01 and for the control sample was 7.92. 

 

Analytical monitoring was conducted by HPLC and MS analysis at all concentration levels. Samples were collected for analysis at study initiation, 24 hour intervals, and then again at termination. Control samples were analysed at initiation and termination only. Results showed that the test material absorbed to the increasing algal biomass. Therefore effect concentrations have been reported in terms of nominal concentrations.

The study fulfilled the validity criteria outlined in the most recent EU guidance (OECD 201, 2006:2011) as detailed below:

• Cell density in the controls should increase by at least a factor of 16 in 72-hours. In the test cell density increased by a factor of 68.17 after 72 hours.


• The coefficient of variation of sectional (daily) growth rates in the controls should be ≤ 35%. In the test this was 11.58%.


• The coefficient of variation of average growth between control replicates should be ≤ 7%. In the test this was 0.4%.

With respect to the inhibitory effect of the test material, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 20.4 - 100.0 mg/L (nominal). Based on the growth rate of the algae the following effect parameters were determined, ErC50 27.77 mg/L, NOEC 6.25 mg/L, and LOEC 12.5 mg/L.

Description of key information

ErC50 27.77 mg/L, NOEC 6.25 mg/L, OECD 201, EU Method C. 3, EPA OPPTS 850. 5400, Sipos 2013c.

Key value for chemical safety assessment

EC50 for freshwater algae:

27.77 mg/L

EC10 or NOEC for freshwater algae:

6.25 mg/L

Additional information

The toxicity of the test material to algae was determined in a growth inhibition study performed with Pseudokirchneriella subcapitata under GLP conditions and in accordance with the standardised guidelines OECD 201, EU Method C.3 and EPA OPPTS 850. 5400. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality as described in Klimisch et al. (1997).

Algal cultures were exposed to the test material at concentrations ranging from 6.25 to 100 mg/L for 72 hours under static freshwater conditions. Cell numbers were manually counted at 24, 48 and 72 hours. Microscopic observations of algal cells were also performed at 24, 48 and 72 hours to detect any abnormal appearance of the algae. The pH at the start for the nominal sample concentration of 100 mg/L was 7.01 and for the control sample was 7.92. 

 

Analytical monitoring was conducted by HPLC and MS analysis at all concentration levels. Samples were collected for analysis at study initiation, 24 hour intervals, and then again at termination. Control samples were analysed at initiation and termination only. Results showed that the test material absorbed to the increasing algal biomass. Therefore effect concentrations have been reported in terms of nominal concentrations.

The study fulfilled the validity criteria outlined in the most recent EU guidance (OECD 201, 2006:2011) as detailed below:

• Cell density in the controls should increase by at least a factor of 16 in 72-hours. In the test cell density increased by a factor of 68.17 after 72 hours.


• The coefficient of variation of sectional (daily) growth rates in the controls should be ≤ 35%. In the test this was 11.58%.


• The coefficient of variation of average growth between control replicates should be ≤ 7%. In the test this was 0.4%.

With respect to the inhibitory effect of the test material, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 20.4 - 100.0 mg/L (nominal). Based on the growth rate of the algae the following effect parameters were determined, ErC50 27.77 mg/L, NOEC 6.25 mg/L, and LOEC 12.5 mg/L.