2-(undec-10-enoylamino)-3-phenylpropanoic acid

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:

2010-04-09 to 2010-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform study without deviations performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

LCa08042

IUPAC Name:

LCa08042

Details on test material:

- Name of test material (as cited in study report): LCA08042
- Lot/batch No.: 0826000002
- Expiration date of the lot/batch: September 16, 2011
- Storage condition of test material: At room temperature (about 20°C), away from direct sunlight

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all tested concentrations
- Sampling method: Treatment samples and control samples were thawed at 25 °C for 30 minutes and shaken manually to obtain homogeneous sample solutions. All aged treatment samples and control samples except sample A141 were filtered (1 µm, GMF) due to the presence of algae.
- Sample storage conditions before analysis: The samples were stored deep-frozen and protected from light until analysis was performed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Due to the low water solubility of the test item, a dispersion with the loading rate of 120 mg/L was prepared by dispersing 120.07 mg of the test item in 1000 mL of test water. The dispersion was supported by ultrasonic treatment for 45 minutes and intense stirring by a magnetic stirrer over 2 hours in the dark, to dissolve a maximum amount of the test item in the dispersion.
The stirring period of 2 hours was chosen according to the results of a pre-experiment (without GLP). After the stirring period, the dispersion of the test item was filtered through a membrane filter (pore size 0.20 μm). The undiluted filtrate was used as the highest concentrated test medium and as stock solution for the preparation of the test media of lower test concentrations. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= start of exposure).
- Eluate: not applicable
- Differential loading: a dispersion with the loading rate of 120 mg/L was prepared
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No auxiliary solvent or emulsifier was used.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Kirchneriella subcapitata KORSIKOV
- Source (laboratory, culture collection): Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): The inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: under test conditions

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no post-observation performed

Test conditions

Hardness:

The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

22 °C

pH:

At the start of the test, the pH measured in the treatments was between 7.7 and 8.5. At the end of the test, pH values of 7.9 to 9.2 were measured (Table 5). The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test.

Dissolved oxygen:

no data

Salinity:

Not particularly stated in the report. Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. The detailed mineral composition of the reconstituted water is described in the original study report.

Nominal and measured concentrations:

nominal concentrations: 2.6, 5.6, 12, 26, 56, 120 mg/L
measured concentrations: 2.37, 5.23, 11.6, 25.2, 53.7, 102 mg/L

Details on test conditions:

MATERIAL
50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.

EXPERIMENTAL CONDITIONS
The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7400 Lux (range: 6800 to 8310 Lux, measured at nine places in the experimental area). The light intensity over the incubation area (measured at nine places in the experimental area) was within ±15% from the average light intensity as recommended by the guideline.

STUDY DESIGN
The selection of the test concentrations was based on the results of range-finding tests (non GLP).
The test design included three replicates per test concentration and six replicates of the control.
The test was started using a nominal algal cell density of 10’000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter, Model ZM).
A static test design was applied. The duration of the test was 72 hours.

DOSAGE
Due to the low water solubility of the test item, a dispersion with the loading rate of 120 mg/L was prepared by dispersing 120.07 mg of the test item in 1000 mL of test water. The dispersion was supported by ultrasonic treatment for 45 minutes and intense stirring by a magnetic stirrer over 2 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.
The stirring period of 2 hours was chosen according to the results of a pre-experiment (without GLP).
After the stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC20, pore size 0.20 µm). The undiluted filtrate was used as the highest concentrated test medium and as stock solution for the preparation of the test media of lower test concentrations. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= start of exposure).

DETERMINATION OF ALGAL BIOMASS
A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
The algal biomass in the samples was determined by measurement of the algal cell density using an electronic particle counter (Coulter Counter, Model ZM). The measurements were performed at least in duplicate.
At the end of the test, a sample was taken from the control and from the undiluted filtrate to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected. Determination of Algal Growth Inhibition and EC Values
Inhibition of algal growth was determined from the following growth parameters:

the specific growth rate (µ)
the yield (Y)

using the following equations:


Specific growth rate (µ):

µi-j = (lnXj – lnXi) / (tj - ti)

where: µi–j: average specific growth rate from time i to j
Xi: biomass at time i
Xj: biomass at time j

The average growth rate over the test duration and the section-by-section growth rates (daily growth rates between the sampling times) were calculated.

Inhibition of growth rate (Ir):

Ir = [ (µC - µTi) / µC ] * 100%

where: Ir: percent inhibition in average specific growth rate
µC: mean value for average specific growth rate in the control group
µT: average specific growth rate for the treatment replicate


Yield (Y):

Y = Xj – X0

where: Y: yield
X0: biomass (nominal value) at the start of the test
Xj: biomass at time j (at the end of the test)

Inhibition of yield (Iy):

Iy = [ (YC - YT) / YC ] * 100%

where: Iy: percent inhibition of yield
YC: mean value for yield in the control group
YT: value for yield for the treatment replicate


Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 78-105

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 9.9-12.2

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

For unknown reasons the undiluted filtrate (120 mg/L) showed a reduced growth inhibition capacity compared to the concentrations 26 and 56 mg/L. This effect could also be observed during the first test (not reported in details) and was the reason for the repetition of the test. As the effects of the dilutions without the undiluted filtrate resulted in a reliable concentration-response relationship, the EC-values were calculated based on the data set without the undiluted filtrate.
The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and biomass) after the test period of 72 hours at 2.6 mg/L and at all higher test concentrations. Thus, the 72 hour LOEC was determined to be 2.6 mg/L and the 72-hour NOEC lower than 2.6 mg/L.
The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing in the undiluted filtrate and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

In the control the biomass increased by a factor of 142 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 5.4%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.1%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

At the start of the test, the pH measured in the treatments was between 7.7 and 8.5. At the end of the test, pH values of 7.9 to 9.2 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test. The water temperature during the test was maintained at 22 °C.

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in September 2009 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.75 mg/L (Harlan study C63678), range of the 72-hour EC50 for the growth rate from 2000 to 2009: 0.71-1.7 mg/L).

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis.

For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Williams t-tests.

Any other information on results incl. tables

Table 2: Biomass of Algaeafter incubation with LCA08042, mean±SD of three replications per concentration (control: 6 replications)

Treatment / Dilution	Nominal test item concentration (mg/L)	Density of algal cell (10^4cells/mL) after
		24 hours	48 hours	72 hours
Control	Control	4.8±0.2	25.4±1.0	142.3±8.3
1:47	2.6	4.7±0.3	24.6±0.7	129.7±1.2
1:22	5.6	4.4±0.1	20.3±0.5	103.2±1.6
1:10	12	4.3±0.2	18.9±0.7	58.2±4.8
1:4.7	26	4.6±0.1	17.9±0.3	38.5±2.0
1:2.2	56	4.3±0.2	16.7±0.7	19.5±1.5
Undiluted filtrate	120	3.7±0.2	18.2±0.3	69.5±5.2

At the start of the test, 1.0 x 104 algal cells/mL were inoculated.

 

 

Table 3: Average Growth Rates(µ)of Algae after incubation with LCA08042, mean±SD of three replications per concentration (control: 6 replications)

Treatment / Dilution	Nominal test item concentration	Average growth rate µ (day-1) and inhibition of µ (Ir)
		0-24 h		0-48 h		0-72 h
	(mg/L)	µ	Ir(%)	µ	Ir(%)	µ	Ir(%)
Control	---	1.56	0.0	1.62	0.0	1.65	0.0
1:47	2.6	1.55	1.2	1.60	1.1	1.62 *	1.8
1:22	5.6	1.48 *	5.3	1.51 *	6.9	1.55 *	6.5
1:10	12	1.47 *	6.3	1.47 *	9.2	1.35 *	18.0
1:4.7	26	1.52 *	2.7	1.44 *	10.9	1.22 *	26.4
1:2.2	56	1.46 *	6.8	1.41 *	13.0	0.99 *	40.1
Undiluted filtrate	120	1.31 *	16.4	1.45 *	10.4	1.41 *	14.5

* mean value statistically significant lower than in the control (according to Williams t-test, one-sided, p < 0.05)

 

 

Table 4:Yield(Y)

Treatment / Dilution	Nominal test item concentration	Yield Y (x 10^4) and inhibitionof Y (Iy)
		0-24 h		0-48 h		0-72 h
	(mg/L)	Y	Iy(%)	Y	Iy(%)	Y	Iy(%)
Control	---	3.8	0.0	24.4	0.0	141.3	0.0
1:47	2.6	3.7	2.2	23.6	3.6	128.7 *	8.9
1:22	5.6	3.4 *	10.1	19.3 *	20.9	102.2 *	27.7
1:10	12	3.3 *	11.9	17.9 *	26.8	57.2 *	59.5
1:4.7	26	3.6 *	5.3	16.9 *	31.0	37.5 *	73.5
1:2.2	56	3.3 *	12.8	15.7 *	35.7	18.5 *	86.9
Undiluted filtrate	120	2.7 *	28.6	17.2 *	29.7	68.5 *	51.6

* mean value statistically significant lower than in the control (according to Williams t-test, one-sided, p < 0.05)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the classification criteria according to „Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008“(CLP), classification shall be based on EC50(growth rate). As the EC50(growth rate) for test item LCA08042
of 89 mg/l falls within the bracket > 10 to ≤ 100 mg/l, thus in OECD-GHS Aqu. Acute 3 would apply, a category not being enforced thru CLP within the EU, though. Aqu. Chron. 3 (CLP) would be applicable to LCA0804 if ever the substance is not readily degradable and/or the experimentally BCF ≥ 500 (or log Kow ≥ 4.0). R52/53 (former DSD regulation) would be applicable to LCA0804 if ever the substance is not readily degradable and/or the experimentally BCF ≥ 100 (or log Kow ≥ 3.0). Note that in both regulations, CLP and DSD, classification for chronic aquatic toxicity would not apply if NOEC > 1.0 mg/l. This point however cannot be proven by this study, which only revealed NOEC < 2.6 mg/l.

Executive summary:

The influence of the test item LCA08042 on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72‑hour static test according to OECD Guideline 201 (2006), the EU Commission Directive 92/69/, C.3 (1992) and the Commission Regulation (EC) No 440/2008, C.3 under GLP. 

A dispersion of the test item was prepared in the test water and stirred for 2 hours in order to dissolve a maximum amount of the test item in the dispersion. After the stirring period, the dispersion was filtered through a membrane filter and the undiluted filtrate (saturated solution) was used as highest test concentration and as stock solution for the preparation of the lower concentrated test media. 

The following treatments were tested: nominal test item concentrations of 2.6, 5.6, 12, 26, 56 and 120 mg/L.

 The measured concentrations of the test item in the test media were between 85 and 97% of the nominal values at the start of the test.During the test period of 72 hours,the measured test item concentration of the undiluted filtrate (120 mg/L) decreased to 1.0% of nominal, the measured concentrations of all dilutions were belowlimit of quantification (LOQ_ana:0.828 mg test item/L). Additionally, the medium of the undiluted filtrate was incubated over the test duration but without algae. The concentration of this test item sample at the end of the test was 86% of nominal and 100% of initially measured. Thus, the losses in the test media with algae during the test period were considered to be due to adsorption of the test item onto the algae.

For unknown reasons the undiluted filtrate (120 mg/L) showed a reduced growth inhibition capacity compared to the concentrations 26 and 56 mg/L. This effect could also be observed during the first test and was the reason for the repetition of the test. As the effects of the dilutions without the undiluted filtrate resulted in a reliable concentration-response relationship, the EC-values were calculated based on the data set without the undiluted filtrate (120 mg/L).

 

The biological results were related to the nominal concentrations (without undiluted filtrate):

 

Parameter	Growth rate	Yield
(0-72 h)
EC10  (mg/L)	7.2	2.2
95% confidence interval	6.0 – 8.4	1.7 – 2.8
EC20  (mg/L)	17	3.9
95% confidence interval	15 – 19	3.2 – 4.5
EC50  (mg/L)	89	11
95% confidence interval	78 – 105	9.9 – 12.2
NOEC (mg/L)	<2.6	<2.6
LOEC (mg/L)	2.6	2.6

Based on the classification criteria according to „Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008“, classification shall be based on EC50(growth rate). As the EC50(growth rate) for test item LCA08042

of 89 mg/l falls within the bracket > 10 to ≤ 100 mg/l, thus in OECD-GHS Aqu. Acute 3 would apply, a category not being enforced thru CLP within the EU, though. Aqu. Chron. 3 (CLP) would be applicable to LCA0804 if ever the substance is not readily degradable and/or the experimentally BCF ≥ 500 (or log Kow ≥ 4.0). R52/53 (former DSD regulation) would be applicable to LCA0804 if ever the substance is not readily degradable and/or the experimentally BCF ≥ 100 (or log Kow ≥ 3.0). Note that in both regulations, CLP and DSD, classification for chronic aquatic toxicity would not apply if NOEC > 1.0 mg/l. This point however cannot be proven by this study, which only revealed NOEC < 2.6 mg/l.