Branched and linear octadecanoic acid, esters with glycerol oligomers

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from similar mixture/product

Adequacy of study:

key study

Study period:

28 Jan - 31 Jan 2013

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Gewerbeaufsicht in Niedersachsen, Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 6.4, 16.0, 40.0, 100, 250 mg/L
- Sampling method: for the test item analysis separate replicates without algae were prepared and incubated under test conditions
- Sample storage conditions before analysis: not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of Water Accommodated Fractions for the exposure to test organisms. For each loading level, an appropriate amount of the test substance was weighed out and applied onto a glass slide. The glass slide with the test substance was inserted into a brown glass flask with an appropriate amount of dilution water. These dispersions were stirred for ≥ 72 h with 1100 rpm at room temperature. After a separation phase of 1 h, the aqueous phase/Water Accommodated Fraction was removed by siphoning (from the approximate centre of the glass flask) to give the loading rate Water Accommodated Fraction.
- Eluate: no
- Differential loading: yes
- Controls: yes, dilution water
- Evidence of undissolved material: not specified, removal of particulate matter by sedimentation

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: Fresh stocks are prepared monthly on Z-Agar. Light intensity 35-70 μE m-2 * s-1 for 24 h per day

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

21.5 - 22.5 °C (mean: 22 °C)

pH:

7.58 - 7.99 (treatments, 0 h); 7.92 - 8.16 (treatments, 72 h); 8.10 (control, 0 h); 8.21 (control, 72 h)

Nominal and measured concentrations:

nominal: 0, 6.40, 16.0, 40.0, 100, 250 mg/L
measured: < 2.00, < 2.00, < 2.00, 11.8 / 7.62 (0 h / 72 h), 5.38 / 3.04 (0 h / 72 h), 3.38 / 2.45 (0 h / 72 h) mg/L TOC

Details on test conditions:

TEST SYSTEM
- Type: closed with cotton wool plugs
- Material, size, headspace, fill volume: sterile Erlenmeyer flask; 250 mL, headspace = 150 mL, fill volume = 100 mL
- Aeration: continuously shaken
- Initial cells density: 4332 cells/mL
- Control end cells density: 776146 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no, nutrient medium Z, according to Lüttge et al. 1994
- Detailed composition if non-standard medium was used: not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guideline
- Total organic carbon: < 2.00 mg/L
- Culture medium different from test medium: yes
- Intervals of water quality measurement: pH was measured at the beginning and end of the test; temperature was measured continuously; light intensity was measured prior to the start of the test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: Mean = 71.3 μE * m-2 * s-1, Coeff. of variation = 5.13 %, n = 31

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: yes, chlorophyll-a-fluorescence, excitation 436 nm, emission 685 nm
- Microscopical observations of cells (unusual cell shape, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: yes
- Test concentrations: control, 2.56, 6.4, 16.0, 40.0, 100 mg/L
- Results used to determine the conditions for the definitive study: yes, at the highest loading of 100 mg/L test substance (corresponding to a TOC of 6.15 mg/L at the test start) the effect on growth rate was low.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (29 Oct - 01 Nov 2012)

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: yes, at nominal test substance loading concentrations 6.4 and 250 mg/L
- 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: yes

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50 (0 - 72 h): Current study: 0.631 mg/L, 95 % confidence interval: 0.583 - 0.687 mg/L; Valid Range (average ± 3 x SD): 0.682 ± 0.452 mg/L
- EyC50 (0 - 72 h): Current study: 0.256 mg/L, 95 % confidence interval: 0.247 - 0.265 mg/L; Valid Range (average ± 3 x SD): 0.314 ± 0.144 mg/L

SD = Standard deviation
ErC50= The loading concentration of the test substance with 50 % inhibition effects (at p = 0.05) derived from the growth rate when compared to the control (calculated by non-linear regression).
EyC50= The loading concentration of the test substance with 50 % inhibition effects (at p = 0.05) derived from the yield when compared to the control (calculated by non-linear regression).

Reported statistics and error estimates:

Descriptive statistics and statistical analyses were prepared with Excel (Microsoft Corp.), SigmaPlot (SPSS Inc.) and GraphPad Prism (GraphPad Software Inc.).

EC VALUES AND STATISTICAL ANALYSES
- EC10, EC20 and EC50 values of the growth rate and yield inhibition after 72 h were not calculated due to the results.

NOEL and LOEL VALUES
- NOEL and LOEL were determined by calculation of statistical significance of growth rate and yield: First a Normality test (Shapiro-Wilk Test) and an Equal Variance test (Levene Median) were done and both yielded p-values of 0.05. Subsequently, a standard One Way Analysis of Variance (ANOVA) and Dunnett´s Test were used.
- The α-value (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05.

Analysis of total organic carbon (TOC) content in test solutions and control at the start and end of the exposure showed that the carbon content in the aqueous phase is not related to the nominal loadings up to 250 mg/L. However, above this concentration no further increase of the carbon content was observed. All effect values are based on the nominal loadings of the test item.

Table 1: Evaluation after 72 h. Statistically significant differences of growth rates and yield compared to control values are marked (+).

Nominal test item loading [mg/L]	Replicate n°	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [%]	Inhibition of yield [%]
250	1	1.79	-3.77	935260	-21.2
	2	1.85	-6.92	1101599	-42.7
	3	1.79	-3.72	932649	-20.8
	Mean	1.81 (+)^#	-4.80	989836 (+)^#	-28.3
100	1	1.71	1.22	720991	6.58
	2	1.78	-2.83	890632	-15.4
	3	1.72	0.44	751120	2.68
	Mean	1.74	-0.39	787581	-2.04
40.0	1	1.67	3.56	638183	17.3
	2	1.71	0.91	732785	5.06
	3	1.65	4.27	614941	20.3
	Mean	1.68	2.91	661970	14.2
16.0	1	1.60	7.37	522919	32.3
	2	1.65	4.76	599562	22.3
	3	1.62	6.45	548833	28.9
	Mean	1.62 (+)	6.19	557105 (+)	27.8
6.4	1	1.82	-5.24	1009387	-30.8
	2	1.85	-7.31	1124652	-45.7
	3	1.81	-4.97	995298	-29.0
	Mean	1.83 (+)^#	-5.84	1043112 (+)^#	-35.2
control	1	1.76		832638
	2	1.75		811095
	3	1.76		856147
	4	1.68		659129
	5	1.73		775777
	6	1.70		696098
	Mean	1.73		771814

Negative inhibitions= increase of growth

#significance due to stimulation

Table 2:Section-by-Section and Average Specific Growth Rates of the Control Group (0 – 72 h).

	Replicate n°	Specific growth rate [d^-1] section-by-section			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
Control		0 – 24 h	24 – 48 h	48 – 72 h
	1	1.58	1.89	1.80	1.76	0.157	8.93	17.6
	2	1.44	2.08	1.72	1.75	0.320	18.3
	3	1.59	1.79	1.91	1.76	0.163	9.24
	4	1.25	2.13	1.65	1.68	0.442	26.4
	5	1.51	1.88	1.81	1.73	0.196	11.3
	6	1.16	2.23	1.69	1.70	0.534	31.5
				Mean	1.73
				SD ±	0.03
				CV [%]	2.00

CV = Coefficient of variation

SD = Standard deviation

 

Table 3.Total organic carbon (TOC) Content of the Test Media at Test Start and Test End.

Nominal test item loading [mg/L]	TOC [mg/L]
	Test start (without algae), 0 h	Test end (without algae), 72 h
250	3.38	2.45
100	5.38	3.04
40	11.8	7.62
16	<2.00	< 2.00
6.4	<2.00	< 2.00
Control	< 2.00	< 2.00

 

Validity criteria fulfilled:

yes

Description of key information

ErL10 (72 h), ErL50 (72 h): > 250 mg/L (nominal, OECD 201); read-across

NOELR (72 h): ≥ 250 mg/L (nominal, OECD 201); read-across

Additional information

There are no studies available investigating the effects of the target substance Polyaldo 2-1-IS (CAS 73296-86-3) on freshwater algae. However, there is one GLP study available for the main constituent Di(isooctadecanoic) acid, diester with oxydi(propanediol) (CAS 67938-21-0), which is considered a reasonable read-across substance to cover this endpoint adequately (see read-across justification in IUCLID section 13).

The study with the source substance Di(isooctadecanoic) acid, diester with oxydi(propanediol) was conducted according to OECD 201 (GLP) and assessed the effect of the substance on the growth rate and yield of freshwater algae. The unicellular freshwater green algae Desmodesmus subspicatus was exposed to five loading rates for 72 h in controlled conditions and continuous illumination. Since the test item is poorly soluble in water, water accommodated fractions (WAF) with nominal loadings ranging from 6.4 to 250 mg test item/L were prepared by continuous stirring and removal of particulate matter by sedimentation. The presence of the test item was analytically verified at the start and the end of exposure by TOC analysis. Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities at test termination. No inhibition of growth was recorded and the derived ErL50 and ErL10 (72 h) were > 250 mg/L (nominal). The corresponding NOELR and LOELR values were ≥ 250 mg/L (nominal) and > 250 mg/L (nominal), respectively.

Based on the available result from one structurally related source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which is a main constituent of the target substance, it can be concluded that the effects of Reaction product of Isooctadecanoic acid, ester with oxybis[propanediol] (CAS 73296-86-3) will be in the same range.