Fatty acids, coco, esters with 1,3-butanediol

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:

30 Oct - 02 Nov 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006, corrected 28 Jul 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Secrétariat général du GIPC, Ivry-sur-Seine, Cedex, France (15 Nov 2018)

Analytical monitoring:

yes

Remarks:

GC-MS/MS

Details on sampling:

- Concentrations: Control, 0.95, 3.05, 9.76, 31.30 and 100 mg/L (after 0 and 72 h)
- Sample storage conditions before analysis: The samples were immediately analysed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was poorly soluble under the test conditions. Therefore, the test solutions were prepared as Water Accommodated Fractions (WAFs) according to OECD guideline 23. The WAFs were prepared individually. Respective amounts of the test item were directly added to the test medium and mixed for 24 h to achieve an equilibrated concentration of dissolved and dispersed or emulsified components in the aqueous phase.
- Differential loading: Yes
- Controls: Test medium only, without test item.
- Evidence of undissolved material: Since the test solutions were not filtered, the test solutions were therefore emulsions of the test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green alga
- Strain: CCAP 1405/1
- Source: Culture Collection of Algae and Protozoa, CCAP
- Age of inoculum: A pre-culture was prepared from the stock culture and maintained in an exponential growth by successive transfers every 3 - 4 d.
- Method of cultivation: The algae were cultured in OECD medium, at 23 ± 2 °C under continuous illumination with intensities in the range of 4440 - 8880 lux

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.56 - 22.75 °C

pH:

7.90 (OECD medium)
0 h: 7.90 (control), 7.90 - 8.00 (treatments)
72 h: 8.10 (control), 7.90 - 8.10 (trreatments)

Nominal and measured concentrations:

Control, 0.95, 3.05, 9.76, 31.30 and 100 mg/L (nominal loading rates for WAFs, see field any other information on results incl. tables for analytical results)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass Erlenmeyer flasks filled with 80 mL test solution and closed with air-permeable polyether foam stoppers.
- Initial cell density: 5000 cells/mL
- Control end cell density: 893383 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Algal stock culture: A stock culture was maintained on sloped agar tubes at 4 °C and regularly transferred to fresh medium.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium was reconstituted with chemical reagents and ultra-pure water.
- Culture medium different from test medium: Culture medium same as test medium
- Intervals of water quality measurement: After 0 and 72 h

OTHER TEST CONDITIONS
- Adjustment of pH: 7.90 (OECD medium)
- Photoperiod: Continuous
- Light intensity: 8120 - 8250 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell biomass was measured every day using an electronic particle counter
- Other: The test was performed in a thermostatic chamber. The flasks were maintained under continuous orbital shaking and the flasks were repositioned each day to minimize differences.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.2
- Range finding study: Yes, a non-GLP range-finder test was performed, test concentrations: 0.02, 0.2, 2, 20 mg/L (nominal loading rate, WAF).
- Results used to determine the conditions for the definitive study: Yes (54.55% inhibition of growth rate at a nominal loading rate of 20 mg/L (WAF))

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

16.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence limit: 15.58 - 18.65 mg/L

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

6.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% confidence limit: 5.76 - 8.23 mg/L

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities: None
- Any stimulation of growth found in any treatment: Stimulation of growth was observed at the nominal loading rate of 0.90 and 3.05 mg/L.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test solutions were emulsions of the test item.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes (dating 18 May 2018)
- ErC50 (72 h): 1.07 mg/L, 95% confidence limits: 0.94 - 1.2 mg/L

Reported statistics and error estimates:

The EC10 and EC50 were calculated according to Regtox® Macro ("Vindimian"), using the Hill model. The model used in this macro is written in the form proposed by Duggleby in 1981. The confidence intervals on the parameters are estimated by a bootstrap simulation which is entirely non-parameteric and is considered to be well adapted to non-linear models (Efron 1990). The NOEC and LOEC were calculated by R software.

VALIDITY CRITERIA

The study fulfilled the validity criteria defined by the guideline (Table 1) and is thus considered valid and reliable.

 

Table 1: Validity criteria for OECD 201.

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.	Actual value: 179	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%	Actual value: 34.95%	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.	Actual value: 4.30%	Yes

 

ANALYTICAL RESULTS

The analytical findings are summarized in Table 2.

 

Table 2. Analytical results

		Nominal loading rate [mg/L]
Time	Analyte	Control	0.95	3.05	9.76	31.30	100
0 h	[mg monoester/L]	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	100
	[mg diester/L]	< LOQ	< LOQ	< LOQ	< LOQ	1.52	5.21
	[mg diester2/L]	< LOQ	< LOQ	< LOQ	< LOQ	1.05	0.53
	[mg diester3/L]	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
	[mg diester4/L]	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
48 h	[mg monoester/L]	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
	[mg diester/L]	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
	[mg diester2/L]	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
	[mg diester3/L]	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ
	[mg diester4/L]	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ

Limit of Quantification (LOQ) = 3.02 mg/L for monoester

LOQ = 0.52 mg/L for diester1

LOQ = 0.73 mg/L for diester2

LOQ = 0.79 mg/L for diester3

LOQ = 0.74 mg/L for diester4

 

BIOLOGICAL RESULTS

Biological findings are summarized in Table 3, Table 4 and Table 5.

 

Table 3. Results in control replicates.

	Number of cells [cells/mL]				Specific growth rate			Growth rate [day^-1]	CV growth [%]	Yield [cells/mL]	CV yield [%]
	0 h	24 h	48 h	72 h	0 – 24 h	24 – 48 h	48 – 72 h	72 h	72 h	72 h	72 h
Control	5000	21444	170400	1113000	1,456	2,073	1,877	1,802	4.3	1108000	23.0
	5000	22514	169300	1170000	1,505	2,018	1,933	1,818		1165000
	5000	21942	151300	898500	1,479	1,931	1,781	1,730		893500
	5000	9752	124400	727100	0,668	2,546	1,766	1,660		722100
	5000	10059	92680	728100	0,699	2,221	2,061	1,660		723100
	5000	10240	137000	723600	0,717	2,594	1,664	1,658		718600
Mean	5000	15992	140847	893383	1,087	2,230	1,847	1,722		888383

Table 4. Results in treatments.

Nominal loading rate [mg/L]	Cell number [cells/mL]				Growth rate [day^-1]	CV growth [%]	Growth rate inhibition [%] compared to control	Yield [cells/mL]	CV yield [%]	Yield inhibition [%] compared to control
	0 h	24 h	48 h	72 h	72 h	72 h	72 h	72 h	72 h	72 h
0.900	5000	10165	171900	1265000	1.844	4.1	-7.14	1260000	22.8	-41.83
	5000	12084	125900	905500	1.733		-0.67	900500		-1.36
	5000	12550	145400	843600	1.709		0.70	838600		5.60
Mean	5000	11600	147733	1004700	1.762		-2.37	999700		-12.53
3.070	5000	22440	153100	1193000	1.825	7.3	-6.01	1188000	34.4	-33.73
	5000	25504	154900	1371000	1.871		-8.70	1366000		-53.76
	5000	22218	86320	662200	1.629		5.39	657200		26.02
Mean	5000	23387	131440	1075400	1.775		-3.10	1070400		-20.49
10.02	5000	10441	48840	432000	1.486	8.6	13.66	427000	37.2	51.94
	5000	4834	47200	268200	1.327		22.89	263200		70.37
	5000	7505	10200	217900	1.258		26.91	212900		76.04
Mean	5000	7593	35413	306033	1.357		21.16	301033		66.11
31.30	5000	4500	6000	11000	0.263	20.4	84.73	6000	31.8	99.32
	5000	14913	6500	13500	0.331		80.77	8500		99.04
	5000	0	7000	16500	0.398		76.88	11500		98.71
Mean	5000	6471	6500	13667	0.331		80.79	8667		99.02
100	5000	2500	2000	4500	0.000	173.2	100.00	0	173.2	100.00
	5000	1500	2500	5500	0.032		98.15	500		99.94
	5000	2000	3500	3500	0.000		100.00	0		100.00
Mean	5000	2000	2667	4500	0.011		99.38	167		99.98

 

Table 5. Effect values (nominal)

NOEC (growth and yield)	3.07 mg/L
LOEC (growth and yield)	10.02 mg/L
ErC10 (72 h)	6.85 mg/L (95% confidence limits: 5.76 – 8.23 mg/L)
ErC50 (72 h)	16.95 mg/L (95% confidence limits: 15.58 – 18.65 mg/L)
EyC10 (72 h)	4.86 mg/L (95% confidence limits: 1.45 – 7.23 mg/L)
EyC50 (72 h)	8.08 mg/L (95% confidence limits: 5.14 – 10.45 mg/L)

 

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

ErL10 (72 h) = 6.85 mg/L (nominal loading rate, OECD 201)

ErL50 (72 h) = 16.95 mg/L (nominal loading rate, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

16.95 mg/L

EC10 or NOEC for freshwater algae:

6.85 mg/L

Additional information

One study is available testing the toxicity of the substance towards algae. The study was conducted according to the OECD guideline 201 and GLP standards. Pseudokirchneriella subcapitata was used as the test organism and was exposed to nominal loading rates of 0.95, 3.05, 9.76, 31.30 and 100 mg/L for 72 h. Since the substance fulfils the UVCB criteria and is partly poorly water soluble the test solutions were prepared as water-accommodated fractions (WAF). The test item quantification was performed by GC-MS/MS at 0 and 72 h. The analytical recovery of the constituents in the medium was mostly below the quantification limit. The study resulted into an ErL10 (72 h) = 6.85 mg/L and an ErL50 (72 h) = 16.95 mg/L (nominal loading rates, OECD 201). All validity criteria were met.