Fatty acid chlorides, C8-14 (even numbered), reaction products with glycine

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:

2010-10-18 to 2010-10-25

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Samples at test start were taken from an additional replicate. At end of the test samples were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution: 320 mg/L, freshly prepared with dilution water
- Eluate: Dilution water
- Differential loading: test concentrations (nominal): 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 - 320 mg/L (factor √10)
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 23, max.: 24, mean value: 23.5

pH:

Nominal test item concentration, pH-value [mg/L], Start (0 h), End (72 h)
320 8.02 8.11
100 8.02 8.20
32.0 8.01 8.25
10.0 8.01 8.48
3.20 7.97 8.52
1.00 7.98 8.51
0.320 7.96 8.12
Control 8.07 8.33

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"
The measured concentrations of Hostapon SG dried (C12 and C14 fraction) at test start were in the range of 81 - 117% and after 72h in the range of 2 % – 111 % of the nominal values. Recoveries for the samples at test start indicate the accurate preparation of the test solutions.
At test end reduced recoveries (< 80% of nominal value) for the C12 fraction were obtained in the exposure groups with 32 and 100 mg/L test item. Analogous to these findings recoveries were < 80% for the C14 fraction in these exposure groups. These reduced recoveries were not a result of an inaccurate analytical method as confirmed by duplicate analysis (results not shown here but documented in the raw data!) but rather caused by the biotic test system.
All effect values were based on the nominal product concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, current: approximately 4 x 103 cells/mL
- Control end cells density: Mean 706945 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 71.8



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
Cell density was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. The pH-value at the beginning of the test was measured out of one additional replicate from each dilution level and the control. At the end it was measured from one pool per concentration and control. The room temperature was measured continuously by a hygro-thermograph. Light intensity was measured prior to test start. Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.



TEST CONCENTRATIONS
- Range finding study: Yes
The results of the preliminary range finding test are presented below:

Results of the Range Finding Test (0-72 h)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition
[%] Yield Inhibition

[%]
100 48 93
10 8 36
1 3 14
0.1 3 13
0.01 4 17

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

68.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

21.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 17.7-26,4 (95% CI)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

61 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 57.3-65.9 (95% CI)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape, Aggregation of algal cells: At the end of the test the cells of the concentrations 32 - 320 mg/L were agglutinated and partly enlarged and at test concentration of 10 mg/L cells were partly enlarged.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item based on nominal concentrations mg/L, (0-72 h)

Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.589 0.738 ± 0.437
95 % confidence interval 0.535 – 0.652
Yield inhibition
EyC50 0.263 0.328 ± 0.146
95 % confidence interval 0.252 – 0.274
SD = Standard deviation

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value for ANOVA was a=0.05. Normality test failed for yield calculation. No suitable transformation could be found. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 % acc. to HEGER et al (1998). These findings were marked “biologically not significant”.
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Hostapon SG dried (0-72 h)

based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	32.0
LOEC	100
ErC10	31.7 (25.9 – 38.6)
ErC20	47.2 (40.8 – 53.5)
ErC50	89.9 (84.3 – 96.2)
	Inhibition of Yield
NOEC	1.00
LOEC	3.20
EyC10	6.27 (3.29 – 12.3)
EyC20	11.2 (6.55 – 17.9)
EyC50	30.5 (21.5 – 43.4)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
320		1		4069		5085	3898	4217
		2		4069		5301	3215	2507
		3		4069		5309	3349	2484
		Mean		4069		5232	3487	3069
100		1		4069		8812	22143	33606
		2		4069		9473	23453	44453
		3		4069		9047	21205	39671
		Mean		4069		9111	22267	39243
32.0		1		4069		10397	67699	418643
		2		4069		11747	85011	440259
		3		4069		14440	86283	459831
		Mean		4069		12195	79664	439578
10.0		1		4069		9999	78784	498107
		2		4069		15527	80618	473943
		3		4069		16193	88817	549963
		Mean		4069		13906	82740	507338
3.20		1		4069		15165	80545	591179
		2		4069		16353	100627	612683
		3		4069		17907	109559	665547
		Mean		4069		16475	96910	623136
1.00		1		4069		16025	117267	777239
		2		4069		16969	138038	749323
		3		4069		19455	130231	903267
		Mean		4069		17483	128512	809943
0.320		1		4069		14911	118973	817839
		2		4069		17755	119950	695395
		3		4069		18063	140779	744199
		Mean		4069		16910	126567	752478
Control		1		4069		14146	108310	658295
		2		4069		16462	149479	857571
		3		4069		19699	127661	778835
		4		4069		17377	112093	664091
		5		4069		16540	104530	626571
		6		4069		20572	124934	656307
		Mean		4069		17466	121168	706945

Evaluation after 72 h

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
320	1		0.012	99.3		148	100
	2		-0.161	100		-1562	100
	3		-0.165	100		-1585	100
	Mean	(+)	-0.105	100	(+)	-1000	100
100	1		0.704	59.0		29537	95.8
	2		0.797	53.6		40384	94.3
	3		0.759	55.8		35602	94.9
	Mean	(+)	0.753	56.1	(+)	35174	95.0
32.0	1		1.55	10.0		414574	41.0
	2		1.56	9.07		436190	37.9
	3		1.58	8.22		455762	35.2
	Mean	(+)*	1.56	9.11	(+)	435509	38.0
10.0	1		1.60	6.67		494038	29.7
	2		1.59	7.64		469874	33.2
	3		1.64	4.75		545894	22.3
	Mean	(+)*	1.61	6.35	(+)	503269	28.4
3.20	1		1.66	3.35		587110	16.5
	2		1.67	2.65		608614	13.4
	3		1.70	1.05		661478	5.89
	Mean	(-)	1.68	2.35	(-)^1	619067	11.9
1.00	1		1.75	-1.97		773170	-10.0
	2		1.74	-1.26		745254	-6.03
	3		1.80	-4.88		899198	-27.9
	Mean	(-)	1.76	-2.70	(-)	805874	-14.7
0.320	1		1.77	-2.95		813770	-15.8
	2		1.71	0.19		691326	1.64
	3		1.74	-1.12		740130	-5.30
	Mean	(-)	1.74	-1.29	(-)	748409	-6.48
Control	1		1.70			654226
	2		1.78			853502
	3		1.75			774766
	4		1.70			660022
	5		1.68			622502
	6		1.69			652238
	Mean		1.72			702876

*) biologically not significant ( 10 %)

1) biologically significant

negative inhibitions = increase of growth

Validity criteria fulfilled:

yes

Remarks:

The cell growth increased 174-fold after 72 h in the control (required: at least 16-fold). The temperature during the test was in the range of 21 – 24 °C. The difference from the mean was within ± 2 °C: min.: 23 °C, max.: 24 °C mean value: 23.5 °C.

Conclusions:

In this study Hostapon SG dried was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 32.0 and 1.00 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 100 and 3.20 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 h were 89.9 (84.3 – 96.2) and 30.5 (21.5 – 43.4) mg/L, respectively.
All effect levels are given based on nominal concentrations of Hostapon SG dried.Values based on a.i. basis are given in the result section.

Executive summary:

The toxicity of Hostapon SG dried (batch number: EK 10/101) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from October 18 to 25, 2010 with the definitive exposure phase from October 18 to 21, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately 4 x 10³cells/mL. Based on a preliminary test 7 concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.320 - 1.00 - 3.20 - 10.0 - 32.0 - 100 - 320 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Hostapon SG dried were analysed at all concentration levels after 0 and 72 h via LC-MS/MS analysis. The measured concentrations of Hostapon SG dried at test start were in the range of 105 – 117 % (C₁₂-chain) and 81 – 110 % (C₁₄-chain) of the nominal values. At the end of the test Hostapon SG dried gave recoveries of 2 - 119 % (C₁₂-chain) and 31 – 106 % (C₁₄-chain) of the nominal values. All effect values are given based on the nominal concentrations of the test item as it contains different C-chains of the N-Cocoyl glycine sodium salt, and toxicity cannot be attributed to any single compound. All effect values are given based on nominal concentrations of the test item.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Hostapon SG dried(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	32.0
LOEC	100
ErC10	31.7 (25.9 – 38.6)
ErC20	47.2 (40.8 – 53.5)
ErC50	89.9 (84.3 – 96.2)
	Inhibition of Yield
NOEC	1.00
LOEC	3.20
EyC10	6.27 (3.29 – 12.3)
EyC20	11.2 (6.55 – 17.9)
EyC50	30.5 (21.5 – 43.4)

Description of key information

In this study Hostapon SG dried was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 32.0 and 1.00 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 100 and 3.20 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 h were 89.9 (84.3 – 96.2) and 30.5 (21.5 – 43.4) mg/L, respectively.

All effect levels are given based on nominal concentrations of Hostapon SG dried.Values based on a.i. basis are given in the result section.

Key value for chemical safety assessment

EC50 for freshwater algae:

61.6 mg/L

EC10 or NOEC for freshwater algae:

21.7 mg/L