Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

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:

05 Dec. 2016 to 06 Jan. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Regulation EU No. 2016/266 amending Regulation EC No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBITION TEST,” adopted 07. December 2015

Deviations:

yes

Remarks:

See "Any other information" for details

Principles of method if other than guideline:

OECD guidance document no. 23, GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, adopted 14. Dec. 2000

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Not specified

Vehicle:

no

Details on test solutions:

For the 1st experiment the water-accommodated fractions (WAF) were prepared individually for the test. This was done mixing the nominal loads with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 23 hours. The resulting solution was filtrated through 0.45 μm PTFE filters.
For the 2nd experiment the water-accommodated fraction (WAF) was prepared as stock solution for the test. This was done mixing the nominal load of 100 mg/L with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm PTFE filters. The lower concentrations were prepared by dilution of this stock solution.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Unicellular freshwater green alga.
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae

Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified

Hardness:

Not specified

Test temperature:

22.5 – 22.6 °C

pH:

7.5 - 9.3

Dissolved oxygen:

Not specified

Salinity:

Not applicable.

Conductivity:

Not specified

Nominal and measured concentrations:

The study was performed using 5 concentrations ranging from 1.0 to 100 mg/L (nominal).

Details on test conditions:

CONDUCT OF THE STUDY
1st Experiment
Pre-Culture
Four days prior to the start of each test, an aliquot of the permanent culture was mixed with nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Performance of the Study
For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (1.34 mL) to achieve a cell concentration of 4.9 *103 cells/mL. In this mixture, the pH-value was measured. Additional samples were obtained for analytical determination.
For the blank control, 350 mL nutrient medium was used in lieu of test item solution and mixed with the necessary amount of algal pre-culture (2.35 mL). In this mixture, the pH value was measured.
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as the starting cell concentration for all replicates.
The test vessels were filled with 45 mL of the respective test solution and incubated open (covered with perforated plastic foil) for 72 hours, and shaken on an orbital shaker. Before the start of incubation, and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (i.e., caused by the exposure to the test item).
The content of DOC in the test vessels was measured at the start and at the end of the test.

Experimental Conditions
Date: 06. – 09. December 2016
Treatments tested: 1.0 / 3.2 / 10 / 32 / 100 mg/L
Number of replicates: 6 replicates for the blank control
3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Temperature: 22.5 – 22.6 °C
Lighting: 4500 Lux
Blank control: deionised water with nutrient medium and algae
Treatments: test solution and algae

2nd Experiment
Pre-Culture
Four days prior to the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours. The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Performance of the Study
For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (1.82 mL) to achieve a cell concentration of 5.8 *103 cells/mL. In this mixture, the pH-value was measured.
Additional samples were obtained for analytical determination.
For the blank control, 350 mL nutrient medium was used instead of test item solution and mixed with the necessary amount of algal pre-culture (3.19 mL). In this mixture, the pH value was measured.
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
The test vessels were filled with 45 mL of the respective test solution and incubated open (covered with perforated plastic foil) for 72 hours, while being kept in constant motion on an orbital shaker. Before the start of incubation and every 24 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The content of DOC in the test vessels was measured at the start and at the end of the test.

Experimental Conditions
Date: 03. - 06. January 2017
Treatments tested: 1.0 / 3.2 / 10 / 32 / 100 mg/L
Number of replicates: 6 replicates for the blank control
3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Temperature: 22.2 - 22.4°C
Lighting: 4500 Lux
Blank control: deionised water with nutrient medium and algae
Treatments: test solution and algae

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate & Yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate & Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate & Yield

Details on results:

In the 1st experiment the treatments were prepared by individual preparation of the water accommodated fraction (WAF). Inhibition in all tested concentrations was within the same range. Therefore, the study was repeated.
In the 2nd experiment the treatments were prepared by dilution of a 100 mg/L (nominal) WAF. This was in accordance with OECD Guidance Document No. 23 where it is stated that serial dilution of a stock WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.
The results of this experiment were used for evaluation of the biological results.
Slight inhibition of algal growth was only observed in the highest concentration of 100 mg/L nominal concentration. However, based on statistical evaluation, inhibition at the nominal concentration 100 mg/L must be stated as not significant and the NOEC must be stated as ≥ 100 mg/L for both endpoints. Whereas for the endpoint yield, the EC 10 must be stated as being within the range of 32 - 100 mg/L.

Results with reference substance (positive control):

The 72h-EC50s of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50s of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Excel ®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1. Biological results are stated using two significant digits.

1st experiment

Cell Numbers

The mean and standard deviations of cell numbers for the blank control, and treatment groups are presented in the table below:

Cell Number/mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	4920	18473	73120	235837
Blank control	SD	0	942	4407	24420
1.0	Mean	4920	19407	131820	165680
1.0	SD	0	1432	6748	15997
3.2	Mean	4920	17680	126027	158647
3.2	SD	0	1134	13694	17348
10	Mean	4920	18873	178233	452887
10	SD	0	896	11784	111566
32	Mean	4920	18893	127213	163127
32	SD	0	367	6301	36931
100	Mean	4920	23033	142487	126360
100	SD	0	7273	10092	2955

SD = Standard Deviation

 

Light Intensity

The table below details light intensity measured during the testing phase:

Light Intensity

	0 h	24 h	48 h	72 h
Light intensity [lux]	4500	4500	4500	4500

 

pH values

In the following table, pH values measured at the start and the end of the test are stated:

pH values

Nominal Concentration in mg/L	0 h	72 h
Blank control	7.5	7.6
1.0	7.5	7.6
3.2	7.5	7.6
10	7.5	8.4
32	7.5	7.6
100	7.5	7.6

 

Microscopical Observations

In the following table, the appearance of the algae at the end of the test is stated:

Microscopical Observations

Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
1.0	Yes
3.2	Yes
10	Yes
32	Yes
100	Yes

 

Analytical Determination

At the start and at the end of each experiment, the content of dissolved organic carbon (DOC) in the test solutions was determined. The content of DOC in all treatments was in the same range as the blank control.

The details of which are given in the following tables:

Measured Concentration IC and TC

Nominal Concentration Test Item	Measured TC t = 0 h	Measured TC t = 72 h	Measured IC t = 0 h	Measured IC t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	7.59	6.51	7.45	6.22
1.0	7.03	6.71	6.82	6.34
3.2	7.14	7.03	7.04	6.64
10	10.44	8.74	7.05	6.70
32	7.88	7.21	7.12	6.18
100	8.00	9.01	7.30	5.94

LOQ (Limit of quantification) TC = 3.17 mg/L

LOQ (Limit of quantification) IC = 1.82 mg/L

 

Measured Concentrations DOC

Nominal Concentration Test Item	Measured DOC (TC-IC) t = 0 h	Measured DOC (TC-IC) t = 72 h	Measured DOC minus blank control t = 0 h	Measured DOC minus blank control t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	0.13	0.30	--	--
1.0	0.21	0.37	0.08	0.07
3.2	0.10	0.39	-0.03	0.09
10	3.38	2.04	3.25	1.74
32	0.76	1.03	0.63	0.73
100	0.70	3.07	0.57	2.77

 

Growth Rate, Yield

From the cell numbers, the Growth Rate μ and the Yield were calculated. The mean, and standard deviations, observed at the end of the test are given in the following table:

Growth Rate μ, Yield

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.29	230917
	SD	0.03	24420
1.0	Mean	1.17	160760
	SD	0.03	15997
3.2	Mean	1.16	153727
	SD	0.04	17348
10	Mean	1.50	447967
	SD	0.08	111566
32	Mean	1.16	158207
	SD	0.08	36931
100	Mean	1.08	121440
	SD	0.01	2955

SD = Standard Deviation

 

Inhibition

The following mean inhibition values were calculated for the Growth Rate μ and the Yield.

Inhibition Values

Nominal Concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
1.0	9.10	30.38
3.2	10.25	33.43
10	-16.50	-93.99
32	9.90	31.49
100	16.03	47.41

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

 

 

2nd experiment

Cell Numbers

The mean, and standard deviations, of cell numbers for the blank control, and treatment groups are presented in the table below:

Cell Number/mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	5820	27757	131383	342993
Blank control	SD	0	2899	18327	96119
1.0	Mean	5820	24100	157127	754480
1.0	SD	0	2279	11867	112007
3.2	Mean	5820	23547	158660	699907
3.2	SD	0	873	11981	174012
10	Mean	5820	27093	172427	900187
10	SD	0	662	6839	69080
32	Mean	5820	24000	156080	642447
32	SD	0	616	4676	45446
100	Mean	5820	25753	130293	272220
100	SD	0	2454	13499	32184

SD = Standard Deviation

 

Light Intensity

The table below details light intensity measured during the testing phase:

Light Intensity

	0 h	24 h	48 h	72 h
Light intensity [lux]	4500	4500	4500	4500

 

pH values

In the following table, the pH values measured at the start and the end of the test are stated:

pH values

Nominal Concentration in mg/L	0 h	72 h
Blank control	8.6	8.2
1.0	8.6	9.2
3.2	8.6	8.9
10	8.6	9.3
32	8.6	9.1
100	8.7	8.2

 

Microscopic Observations

In the following table, the appearance of the algae at the end of the test is stated:

Microscopical Observations

Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
1.0	Yes
3.2	Yes
10	Yes
32	Yes
100	Yes

 

Analytical Determination

At the start and at the end of each experiment, the content of dissolved organic carbon (DOC) in the test solutions was determined. The content of DOC in all treatments was in the same range as the blank control.

Therefore, determination of the biological results was based on the nominal concentrations.

The details of which are given in the following tables:

Measured Concentration IC and TC

Nominal Concentration Test Item	Measured TC t = 0 h	Measured TC t = 72 h	Measured IC t = 0 h	Measured IC t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	0.00	14.41	13.42	14.35
1.0	14.01	0.00	14.00	0.49
3.2	13.82	11.25	14.16	11.24
10	14.04	11.12	14.05	10.75
32	14.02	11.27	14.08	11.60
100	15.99	15.16	13.78	14.91

LOQ (Limit of quantification) TC = 2.81 mg/L

LOQ (Limit of quantification) IC = 1.08 mg/L

 

Measured Concentrations DOC

Nominal Concentration Test Item	Measured DOC (TC-IC) t = 0 h	Measured DOC (TC-IC) t = 72 h	Measured DOC minus blank control t = 0 h	Measured DOC minus blank control t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	0.00	0.06	--	--
1.0	0.01	0.00	0.01	-0.06
3.2	0.00	0.02	0.00	-0.04
10	0.00	0.37	0.00	0.32
32	0.00	0.00	0.00	-0.06
100	2.22	0.25	2.22	0.20

 

Growth Rate, Yield

From the cell numbers, the Growth Rate μ and the Yield were calculated. The mean and standard deviations, observed at the end of the test were measured, and are given in the following table:

Growth Rate μ, Yield

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.35	337173
	SD	0.09	96119
1.0	Mean	1.62	748660
	SD	0.05	112007
3.2	Mean	1.59	694087
	SD	0.09	174012
10	Mean	1.68	894367
	SD	0.03	69080
32	Mean	1.57	636627
	SD	0.02	45446
100	Mean	1.28	266400
	SD	0.04	32184

SD = Standard Deviation

 

Inhibition

The following mean inhibition values were calculated for the Growth Rate μ and the Yield.

Inhibition Values

Nominal Concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
1.0	-20.02	-122.04
3.2	-17.80	-105.85
10	-24.50	-165.25
32	-16.18	-88.81
100	5.11	20.99

Negative inhibition values indicate a stimulation of algal growth compared to the blank control.

 

 

Biological Results of the Reference Study (16102401R301)

The results of the last study with the positive control K2Cr2O7 are presented in the following table. The study was performed under GLP conditions in October 2016.

Parameter	Value	95% confidence interval
72h ErC50	0.80 mg/L	0.78 – 0.83 mg/L
72h EyC50	0.54 mg/L	0.52 – 0.56 mg/L

 

Validity criteria fulfilled:

yes

Conclusions:

The following results for the test item Hatcol 3178 were determined:
NOEC (Growth Rate) ≥ 100 mg/L
NOEC (Yield) ≥ 100 mg/L
LOEC (Growth Rate) > 100 mg/L
LOEC (Yield) > 100 mg/L
EC50 (Growth rate) > 100 mg/L
EC50 (Yield) > 100 mg/L

Executive summary:

Two valid experiments were performed.

The study was performed using 5 concentrations ranging from 1.0 to 100 mg/L (nominal).

Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate μ and the yield were determined from the cell number at the respective observation times.

 

In the 1st experiment the treatments were prepared by individual preparation of the water accommodated fraction (WAF). Inhibition in all tested concentrations was within the same range except the treatment containing 10 mg/L nominal concentration. Therefore, the study was repeated.

 

In the 2nd experiment the treatments were prepared by dilution of a 100 mg/L (nominal) WAF. This was in accordance with OECD Guidance Document No. 23 where it is stated that serial dilution of a stock WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.

The results of this experiment were used for evaluation of the biological results.

Slight inhibition of algal growth was only observed in the highest concentration of 100 mg/L nominal concentration. However, based on statistical evaluation, inhibition at the nominal concentration 100 mg/L must be stated as not significant and the NOEC must be stated as ≥ 100 mg/L for both endpoints. Whereas for the endpoint yield, the EC 10 must be stated as being within the range 32 - 100 mg/L.

 

At the start and at the end of each experiment, the content of dissolved organic carbon (DOC) in the test solutions was determined.

In the 1st experiment the content of DOC in all treatments was low and partly in the same range as the blank control. No dose-response relationship between increasing nominal load and rising DOC was observable.

In the 2nd experiment the content of DOC in all treatments was in the same range as the blank control.

 

However, slight inhibition of algal growth indicates the presence of a very low concentration of dissolved test item in the test solution.

Therefore, determination of the biological results was based on the nominal concentrations.

 

The 72h-EC50s of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

 

The following results for the test item Hatcol 3178 were determined:

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	≥ 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L
Yield	≥ 100 mg/L	> 100 mg/L	Within 32 – 100 mg/L	> 100 mg/L

 

Note: According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.

Description of key information

The following results for the test item Hatcol 3178 were determined:

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	≥ 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L
Yield	≥ 100 mg/L	> 100 mg/L	Within 32 – 100 mg/L	> 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Two valid experiments were performed.

The study was performed using 5 concentrations ranging from 1.0 to 100 mg/L (nominal).

Incubation time (test system Desmodesmus subspicatus) was 72 hours.

 

In the 1st experiment the treatments were prepared by individual preparation of the water accommodated fraction (WAF). Inhibition in all tested concentrations was within the same range except the treatment containing 10 mg/L nominal concentration. Therefore, the study was repeated.

 

In the 2nd experiment the treatments were prepared by dilution of a 100 mg/L (nominal) WAF. This was in accordance with OECD Guidance Document No. 23 where it is stated that serial dilution of a stock WAF may be the only applicable method for chemicals being toxic at concentrations of 1 mg/L or lower.

The results of this experiment were used for evaluation of the biological results.

Slight inhibition of algal growth was only observed in the highest concentration of 100 mg/L nominal concentration. However, based on statistical evaluation, inhibition at the nominal concentration 100 mg/L must be stated as not significant and the NOEC must be stated as ≥ 100 mg/L for both endpoints. Whereas for the endpoint yield, the EC 10 must be stated as being within the range 32 - 100 mg/L.

 

The following results for the test item Hatcol 3178 were determined:

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	≥ 100 mg/L	> 100 mg/L	> 100 mg/L	> 100 mg/L
Yield	≥ 100 mg/L	> 100 mg/L	Within 32 – 100 mg/L	> 100 mg/L