Decanoic acid, mixed esters with heptanoic acid, isovaleric 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:

09. Apr. 2018 to 13. Apr. 2018

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:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth 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

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:

Samples taken from each mixture prepared.

Vehicle:

no

Details on test solutions:

A water-accommodated fraction (WAF) was prepared for the test. This was done by mixing the nominal loads 1 / 3.2 / 10 / 32 / 100 mg/L resp. 1.01 / 3.23 / 10.10 / 32.32 / 101.01 μL/L test item (based on a density of 0.99 g/mL stated in the MSDS from the sponsor) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and stirring moderately for 24 hours. The lower phase was used unfiltered as test solutions.

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:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified.

Hardness:

Not specified

Test temperature:

21.8 – 23.5 °C

pH:

7.7 - 8.4

Dissolved oxygen:

Not specified

Salinity:

Not applicable - freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

1 / 3.2 / 10 / 32 / 100 mg/L nominal concentrations.
The concentrations were based on non GLP pre-tests.

Details on test conditions:

Pre-Culture
Four days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test conditions. 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 (0.363 mL) to achieve a cell concentration of 2.4*103 cells/mL. 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 (0.635 mL).
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.
From these mixtures samples for the analytical determination were taken, afterwards the pH-value was measured.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 ±1 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: 10. – 13. Apr. 2018
Treatments tested: 1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration
The concentrations were based on non GLP pre-tests.
Number of replicates: 6 replicates for the blank control; 3 replicates for each treatment
Vessels: glass flasks, total volume 65 mL
Duration: 72 hours
Lighting: 5000 lux
Temperature: 21.8 – 23.5 °C
Replicates (Blank control): 45 ±1 mL algal medium and algae
Replicates (Treatments): 45 ±1 mL 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.:

10 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.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: grwoth 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: grwoth rate & yield

Details on results:

One valid experiment was performed.
The study was performed using 5 concentrations ranging from 1 to 100 mg/L. 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.
Significant inhibition of algal growth was observed at the following concentrations:
32 – 100 mg/L (nominal)
In treatment 3.2 mg/L, flocculation of the algae or precipitation could be be detected in the form of turbidity. Therefore, measurement of the cell numbers was biased and the calculated inhibition was faulty. Therefore, this treatment is not used for evaluation.
At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.
Since the measured concentrations are in the same very low range as the blank control, the nominal values are used for statistical evaluation. Since the used test solutions were not filtered after stirring, and inhibition of algal growth was observed in the 2 highest concentrations the test item must have been present in the test solution.
Because inhibition of algal growth was only slight, only the range can be specified for the ECx calues, with the exception of the EC10 for the endpoint yield.
The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values 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.
The pH of the blank control should not fluctuate by more than 1.5 units. The change was 0.2 units in the blank control.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Results with reference substance (positive control):

The 72h-EC50 values 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).

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. Since the measured concentration is in the same very low range as the blank control, the nominal values are used for statistical evaluation.

Exact loads for the preparation of the water-accommodated fractions

Nominal concentration in mg/L	Read load in mg/L
1	0.99
3.2	3.17
10	10
32	32
100	100

 

Biological Results of K₂Cr₂O₇

Parameter	Value	95% confidence interval
72 h ErC50	0.90 mg/L	0.77 – 1.1 mg/L
72 h EyC50	0.42 mg/L	0.35 – 0.51 mg/L

 

Cell Number/mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2440	10203	64143	325323
Blank control	SD	0	694	3600	20124
1	Mean	2440	8107	49300	298313
1	SD	0	833	3721	29284
3.2	Mean	2440	7520	36993	181953
3.2	SD	0	1576	6207	29596
10	Mean	2440	9587	59333	310407
10	SD	0	383	2832	18522
32	Mean	2440	10180	57713	286200
32	SD	0	701	1769	6017
100	Mean	2440	8167	43560	235140
100	SD	0	491	7337	36512

Treatment 3.2 mg/L was not used for evaluation due to flocculation or precipitation

SD = Standard Deviation

 

Light Intensity

	0 h	24 h	48 h	72 h
Light intensity [Lux]	5000	5000	5000	5000

 

pH values

Nominal Concentration in mg/L	0 h	72 h
Blank control	7.7	7.9
1	7.7	7.8
3.2	7.8	8.1
10	7.7	8.4
32	7.7	8.3
100	7.7	8.2

 

Microscopical Observations

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

 

Measured Concentrations 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	7.29	6.52	6.84	5.62
1	7.28	6.52	6.69	5.74
3.2	7.44	6.85	7.02	6.10
10	7.42	6.39	7.00	5.06
32	7.67	6.74	7.04	5.89
100	7.80	7.11	7.05	6.12

LOQ (Limit of Quantification) TC = 5.43 mg/L

LOQ (Limit of Quantification) IC = 2.03 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	0.45	0.90	--	--
1	0.59	0.79	0.14	-0.12
3.2	0.42	0.5	-0.02	-0.15
10	0.42	1.33	-0.02	0.43
32	0.63	0.85	0.19	-0.05
100	0.75	1.00	0.30	0.09

 

Calculated Test Item Concentrations

Nominal Concentration Test Item	Calculated Concentration t = 0 h	Calculated Concentration t = 72 h	% of Nominal concentration t = 0 h	% of Nominal concentration t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	--	--	--	--
1	0.2	-0.2	23	-18
3.2	0.0	-0.2	-1	-7
10	0.0	0.7	0	7
32	0.3	-0.1	1	0
100	0.5	0.1	0	0

 

Growth Rate μ, Yield

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72 h) [day^-1]	Yield (0-72 h) [Cell Concentration/mL]
Blank control	Mean	1.63	322883
	SD	0.02	20124
1	Mean	1.60	295873
	SD	0.03	29284
3.2	Mean	1.43	179513
	SD	0.05	29596
10	Mean	1.61	307967
	SD	0.02	18522
32	Mean	1.59	283760
	SD	0.01	6017
100	Mean	1.52	232700
	SD	0.05	36512

Treatment 3.2 mg/L was not used for evaluation due to flocculation or precipitation

SD = Standard Deviation

 

Inhibition Values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72 h)	Yield (0-72 h)
Blank control	0	0
1	1.8	8.4
10	1.0	4.6
32	2.6	12.1
100	6.8	27.9

Treatment 3.2 mg/L was not used for evaluation due to flocculation or precipitation

Validity criteria fulfilled:

yes

Conclusions:

The following results for the test item Hatcol 1570 were determined:
Endpoint NOEC LOEC EC10 EC50
Growth Rate 10 mg/L 32 mg/L > 100 mg/L > 100 mg/L
Yield 10 mg/L 32 mg/L 33.07 mg/L > 100 mg/L

Executive summary:

Determination of the toxicity of Hatcol 1570 against Desmodesmus subspicatus according to OECD 201 resp. EU C.3

 

Findings and Results:

One valid experiment was performed.

The study was performed using 5 concentrations ranging from 1 to 100 mg/L nominal concentration.

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.

Significant inhibition of algal growth was observed at the following concentrations:

32 – 100 mg/L (nominal)

In treatment 3.2 mg/L, flocculation of the algae or precipitation could be detected in the form of turbidity. Therefore, measurement of the cell numbers was biased and the calculated inhibition was faulty. Therefore, this treatment was not used for evaluation.

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

Since the measured concentrations are in the same very low range as the blank control, the nominal values are used for statistical evaluation. Since the used test solutions were not filtered after stirring, and inhibition of algal growth was observed in the 2 highest concentrations, the test item must have been present in the test solution.

Because inhibition of algal growth was only slight, only the range can be specified for the ECx values, with the exception of the EC10 for the endpoint yield.

The 72h-EC50 values 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 1570 were determined:

 

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	10 mg/L	32 mg/L	> 100 mg/L	> 100 mg/L
Yield	10 mg/L	32 mg/L	33.07 mg/L	> 100 mg/L

Description of key information

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	10 mg/L	32 mg/L	> 100 mg/L	> 100 mg/L
Yield	10 mg/L	32 mg/L	33.07 mg/L	> 100 mg/L

 

No toxicity at the limit of solubility in water.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

One valid experiment was performed.

The study was performed using 5 concentrations ranging from 1 to 100 mg/L nominal concentration.

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.

Significant inhibition of algal growth was observed at the following concentrations: 32 – 100 mg/L (nominal)

In treatment 3.2 mg/L, flocculation of the algae or precipitation could be detected in the form of turbidity. Therefore, measurement of the cell numbers was biased and the calculated inhibition was faulty. Therefore, this treatment was not used for evaluation.

Since the used test solutions were not filtered after stirring, and inhibition of algal growth was observed in the 2 highest concentrations, the test item must have been present in the test solution.

Because inhibition of algal growth was only slight, only the range can be specified for the ECx values, with the exception of the EC10 for the endpoint yield.

The following results for the test item Hatcol 1570 were determined:

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	10 mg/L	32 mg/L	> 100 mg/L	> 100 mg/L
Yield	10 mg/L	32 mg/L	33.07 mg/L	> 100 mg/L