Fatty acids, C16-18, esters with diethylene glycol

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:

15 Jun - 22 Jul 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of the Government of teh United Kingdom

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0, 10, 20, 40, 80 and 160 mg/L
- Sample storage conditions before analysis: stored frozen

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Nominal amounts of 20, 40, 80, 160 and 320 mg were each separately added to the surface of 2 L of culture medium to give nominal loading rates of 10, 20, 40, 80 and 160 mg/L. The culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. Stirring was stopped after 23 h and the mixture was allowed to stand for 1 h. Visual observations of the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence the WAF was filtered through a glass wool plug (2-4 cm in length) followed by Postlip filter paper. A wide bore glass tube, covered at the end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approx. 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (75-100 mL discarded) to give the loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of the test item present. An aliquot of 1 L of each of the loading rate WAFs was separately inoculated with algal suspension (6.8 mL) to give the required loading rate WAFs.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

ACCLIMATION
- Acclimation period: Prior to the start master culture was added to approx. 100 mL of the culture medium. The initial cell density was approx. 10E+003 cells/mL. Flasks were plugged with PU foam stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approx. 10E+004 - 10E+005 cells/mL.
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

control: 7.7 - 9.0 (0 - 72 h)
10 mg/L: 7.6 - 9.0 (0 - 72 h)
20 mg/L: 7.4 - 8.9 (0 - 72 h)
40 mg/L: 7.3 - 8.8 (0 - 72 h)
80 mg/L: 7.3 - 8.7 (0 - 72 h)
160 mg/L: 7.3 - 8.7 (0 - 72 h)

Nominal and measured concentrations:

nominal: Control, 10, 20, 40, 80 and 160 mg/L
measured 0 h: < LOQ, 0.00452, < LOQ, 0.00645, 0.0407, 0.0144 mg/L
measured 72 h: < LOQ in all test samples

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks (plugged with polyurethane foam bungs)
- Material, size, headspace, fill volume: glass, 250 mL, headspace: 150 mL, fill volume: 100 mL
- Aeration: constantly shaken at 150 rpm
- Initial cells density: approx. 5E+003 cells/mL
- Control end cells density: 7.54E+005 cells/mL (mean of six replicates)
- 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: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was recorded daily; pH was recorded after 0 and 72 h

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: approx 7000 lux (warm lighting: 380 - 730 nm)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Coulter® Multisizer Particle Counter; measured after 0, 24, 46 and 72 h
- Other: Shape and size of algal cells was microscopically inspected after 72 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 10, 100 mg/L
- Results used to determine the conditions for the definitive study: At a loading rate of 10 mg/L and increase in yield was observed (12%). At 100 mg/L loading rate a decrease in growth rate (14%) and yield (46%) was recorded.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (Study number: 41501180)

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 160 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 160 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 160 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No micro-dispersions were observed after microscopic evaluation.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50: 1.0 mg/L (95% CI: 0.90 - 1.2 mg/L); EyC50: 0.49 mg/L (95% CI: 0.42 - 0.58 mg/L); NOErC: 0.25 mg/L

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett’s test for homogeneity of variance and Dunnett’s multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 h for the control and all test loading rates to determine any statistically significant differences between the test and control groups. Statistical analysis was performed using SAS computer software package.

Given that the toxicity cannot be atributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Table 1: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal loading rate [mg/L]		Growth rate [cells/mL/hour]		Yield [cells/mL]
		0-72 h	% inhibition	0-72 h	% inhibition
Control	R1	0.070	-	7.68E+05	-
	R2	0.072	-	8.59E+05	-
	R3	0.069	-	7.12 E+05	-
	R4	0.071	-	8.33 E+05	-
	R5	0.069	-	7.30 E+05	-
	R6	0.066	-	5.94 E+05	-
	Mean	0.070	-	7.49 E+05	-
	SD	0.002	-	9.55 E+04	-
10	R1	0.069	1	7.03 E+05
	R2	0.067	4	5.99 E+05
	R3	0.069	1	7.00 E+05
	Mean	0.068	2	6.67 E+05	11
	SD	0.001		5.94 E+04
20	R1	0.071	[1]	8.01 E+05
	R2	0.065	7	5.51 E+05
	R3	0.066	6	5.63 E+05
	Mean	0.067	4	6.38 E+05	15
	SD	0.003		1.41 E+05
40	R1	0.067	4	6.37 E+05
	R2	0.065	7	5.18 E+05
	R3	0.063	10	4.49 E+05
	Mean	0.065	7	5.34 E+05	29
	SD	0.002		9.52 E+04
80	R1	0.067	4	5.96 E+05
	R2	0.061	13	3.94 E+05
	R3	0.065	7	5.28 E+05
	Mean	0.064	8	5.06 E+05	32
	SD	0.003		1.03 E+05
160	R1	0.065	7	5.43 E+05
	R2	0.066	6	5.84 E+05
	R3	0.064	9	4.94 E+05
	Mean	0.065	7	5.40 E+05	28
	SD	0.001		4.55 E+04

[Increase in growth as compared to the control]

Table 2: Validity criteria for OECD 201 (2006)

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	The cell concentration of the control cultures increased by a factor of 148 after 72 hours.	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	The mean coefficient of variation for section-by-section specific growth rate for the control cultures was 10%.	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	The coefficient of variation of average specific growth rate for the control cultures over the test period (0 – 72 h) was 3%.	yes

 

Validity criteria fulfilled:

yes

Remarks:

See Table 2 in "Any other information on results incl. tables".

Description of key information

No effects up to the limit of water solubility (OECD 201)

Additional information

One experimental study is available investigating the toxicity of Fatty acids, C16-18, esters with diethylene glycol (CAS 85116-97-8) to freshwater algae. The study was performed according to OECD 201 (GLP; Vryenhoef, 2015). Since the test substance is poorly soluble in water, Water Accommodated Fractions (WAF) of 10, 20, 40, 80 and 100 mg/L (nominal) were prepared. The nominal loading rates were analytically verified by HPLC/MS analysis and resulted in concentrations 0.00452 - 0.0407 mg/L at study initiation and depleted below the limit of quantification at the end of the study after 72 h.
After incubation, inhibition of growth of 2% was recorded at 10 mg/L, 4% at 20 mg/L, 7% at 40 mg/L, 8% at 80 mg/L and 7% at 160 mg/L. Statistical analysis resulted in significant difference between the control and the treatment of 40 mg/L. Thus, the NOELR (72 h) was set to 20 mg/L based on growth rate. However, the inhibition of growth was < 10% in all treatments resulting in an ErL10 (72 h) > 160 mg/L. These effects are not considered to be biologically relevant and the ErL10 was used for the final assessment and classification purposes. This is in line with OECD Guidance on statistical evaluation of ecotoxicological data (OECD, 2006) on the guidance on application of CLP criteria (ECHA, 2015).

In conclusion the test substance will not exhibit toxic effects to aquatic algae up to the limit of water solubility.

 

OECD 2006: Series on Testing and Assessment Number 54, Current approaches in the statistical analysis of ecotoxicity data: a guidance to application. ENV/JM/MONO(2006)18