Fatty acids, tall-oil, 1-methyl-1,2-ethanediyl esters

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

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, corrected 2011)

Deviations:

yes

Remarks:

(the variation of the light intensity exceeded 15% (25-27%). The deviation was considered not to have affected the integrity of the study as the guideline recommended range of individual measurements was met and the validity criteria were fulfilled.)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2009)

Deviations:

yes

Remarks:

(the variation of the light intensity exceeded 15% (25-27%). The deviation was considered not to have affected the integrity of the study as the guideline recommended range of individual measurements was met and the validity criteria were fulfilled.)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

On each sampling occasion, one sample from each level was analysed for TOC. Samples at test end were taken from flasks which contained no algal cells.

Vehicle:

no

Details on test solutions:

REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
For the test media with nominal loading rates of 100, 31.3, 9.77, 3.05 and 0.954 mg/L aliquots of the test material were dispersed in dilution medium. The preparations were stirred for approximately 24 hours in the dark, then left to stand for approximately 24 hours in the dark. An aliquot was then syphoned from a mid-vessel location of each preparation vessel, of which the first litre was discarded, to give the Water Accommodated Fractions (WAF) which were used as test media. The lowest nominal loading rate of 0.298 mg/L was prepared by dilution of an aliquot of the WAF at 0.954 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Three days. Algal cells used for inoculation were in the log phase of growth.
- Method of cultivation: Liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 24°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for three days and used as inoculum for the test.

ACCLIMATION
- Acclimation period: 3 days (pre-culturing)
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3

Test temperature:

22-24 °C

pH:

test start (0 h): 7.4-7.9
test end (72 h): 8.9-9.8

Nominal and measured concentrations:

nominal loading rates: 0.298, 0.954, 3.05, 9.77, 31.3 and 100 mg/L as WAF
mean measured TOC levels (after background correction):
test start:
2.61 and 3.56 mg/L at nominal 31.3 and 100 mg/L;
control mean: 2.76 mg C/L.

test end (72 hours):
1.87 and 3.18 mg/L at nominal 31.3 and 100 mg/L;
control mean: 2.88 mg C/L.

arithmetric mean: 2.21 and 3.37 mg/L at nominal 31.3 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks (autoclaved) containing 100 mL of inoculated test medium and loosly plugged with foam bungs
- Aeration/gaseous exchange: orbital incubator, oscillating at nominal 130 cycles per minute
- Initial cells density: ca. 1x10⁴ cells/mL
- Control end cells density: 198x10⁴ cells/mL
- 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: OECD medium according to guidelines, using filtered, dechlorinated tap water, softened and treated by reverse osmosis before microfiltration and purification (resistivity 18 Megohm/cm)
- Total organic carbon: 2.8 mg C/L
- Ca/Mg ratio: 1:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 4620 to 7340 Lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. Light intensity within the test area was determined each day at four corner positions and in a central position of the random block design. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the test.
The light intensity measured at the corner positions was within ±15% of the mean value over the study period. In the center of the incubator, it ranged between 25-27% of the mean. As the individual measurements were all guideline conform (4440 - 8880 Lux) and the validity criteria were met, this deviation to guideline was not thought to have affected either the validity or integrity of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell densities measured at 0, 24, 48 and 72 hours
- Determination of cell concentrations: haemocytometer (Improved Neubauer)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
inhibition of algal growth after 72 hours was 95 and 17% at 100 and 10 mg/L and not significant at 1.0 mg/L.

Reference substance (positive control):

not specified

Remarks:

Laboratory reference tests using potassium dichromate are performed on a regulary basis but were not reported.

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: results are expressed in terms of loading rates

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: no biologically significant stimulation of growth was observed (see Tables 1 and 2).

At the start of the test, the test media (as WAF) at nominal loading rates of 31.3 and 100 mg/L were hazy. All other conentrations were colourless.

Reported statistics and error estimates:

Statistical analysis was perfomed using SAS 9.1 (SAS Institute 2002), using nominal loading rates. 95% confidence intervals were calculated using the likelihood ratio method. Williams’ test was used to compare each treated group with control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test was used.

In a first main test employing WAFs with nominal loading rates of 0.298, 0.954, 3.05, 9.77, 31.3 and 100 mg/L, growth was inhibited by 20% at 100 mg/L after at 72 hours, with no significant inhibition in at any of the other test groups (except 19% inhibition was observed at 3.05 mg/L). Owing to variation in the results of TOC analysis and the difference in biological data compared with the range finding phase, the test was repeated using the same test concentrations and under the same exposure conditions. The data obtained in the first main test was replicated in the repeat test, suggesting that the findings in the range finding test were not representative and the reason for this was not known. The data from the repeat test are reported.

 

Table 1: Cell densities

 

Nominal loading rates (mg/L)	Replicate number	Cell densities (cells/mL)
		0 hours	24 hours	48 hours	72 hours
Control	R1	10000	53750	313750	2152500
	R2	10000	75000	337500	2105000
	R3	10000	53750	336250	1885000
	R4	10000	71250	305000	1972500
	R5	10000	47500	320000	1847500
	R6	10000	58750	323750	1912500
	Mean	10000	60000	322708	1979167
0.298	R1	10000	53750	317500	1665000
	R2	10000	71250	246250	1815000
	R3	10000	61250	278750	2105000
	Mean	10000	62083	280833	1861667
0.954	R1	11250	51250	327500	1730000
	R2	11250	65000	361250	1835000
	R3	11250	60000	333750	1595000
	Mean	11250	58750	340833	1720000
3.05	R1	12500	55000	312500	1690000
	R2	12500	51250	315000	1747500
	R3	12500	65000	347500	2035000
	Mean	12500	57083	325000	1824167
9.77	R1	11250	70000	316250	1620000
	R2	11250	53750	358750	1927500
	R3	11250	67500	338750	1830000
	Mean	11250	63750	337917	1792500
31.3	R1	10000	42500	316250	1490000
	R2	10000	68750	351250	2027500
	R3	10000	45000	311250	1810000
	Mean	10000	52083	326250	1775833
100	R1	8750	73750	356250	1667500
	R2	8750	95000	397500	2047500
	R3	8750	71250	363750	1835000
	Mean	8750	80000	372500	1850000

 

R_1-R₆        replicate number

 

 

Table 2  Inhibition of growth

 

Parameter	Nominal loading rates (mg/L)	Sample size	Mean	Inhibition (%)	p-value
Growth rate to 72 hours	Control	6	0.0734	0.0
	0.00931	3	0.0725	1.2	0.892^D
	0.0298	3	0.0698	4.9	0.009**^D
	0.0954	3	0.0692	5.8	0.002**^D
	0.305	3	0.0704	4.1	0.030*^D
	0.977	3	0.0718	2.2	0.443^D
	3.13	3	0.0743	-1.2	0.893^D
Growth rate 0 to 24 hours	Control	6	0.0741	0.0
	0.00931	3	0.0758	-2.3	1.000^D
	0.0298	3	0.0687	7.3	0.809^D
	0.0954	3	0.0631	14.9	0.184^D
	0.305	3	0.0720	2.8	0.997^D
	0.977	3	0.0678	8.6	0.695^D
	3.13	3	0.0919	-24.0	0.013*^D
Growth rate 24 to 48 hours	Control	6	0.0706	0.0
	0.00931	3	0.0629	10.9	0.542^D
	0.0298	3	0.0734	-4.0	0.990^D
	0.0954	3	0.0726	-2.8	0.998^D
	0.305	3	0.0697	1.3	1.000^D
	0.977	3	0.0774	-9.6	0.663^D
	3.13	3	0.0644	8.8	0.732^D
Growth rate 48 to 72 hours	Control	6	0.0755	0.0
	0.00931	3	0.0788	-4.4	1.000^W
	0.0298	3	0.0674	10.7	0.067^W
	0.0954	3	0.0718	5.0	0.067^W
	0.305	3	0.0695	8.0	0.067^W
	0.977	3	0.0703	6.9	0.067^W
	3.13	3	0.0667	11.7	0.007**^W
Yield to 72 hours	Control	6	1969167	0.0
	0.00931	3	1851667	6.0	0.365^W
	0.0298	3	1708750	13.2	0.197^W
	0.0954	3	1811667	8.0	0.197^W
	0.305	3	1781250	9.5	0.197^W
	0.977	3	1765833	10.3	0.197^W
	3.13	3	1841250	6.5	0.197^W

 

p values are for the comparison with Control using Williams' test (W) and Dunnett's test (D)

*p< 0.05,  **p< 0.01,  ***p< 0.001

Note: Negative values indicate an increase in algal growth above that of Control growth

 

Control cultures:

-cell concentration increased by a factor of 198;

-the mean coefficient of variation for section by section daily growth rates in the control cultures was 8.9%;

- the coefficient of variation for the average specific growth rates of the control cultures was 1.17% during the 72 hour exposure period.

 

Validity criteria fulfilled:

yes

Remarks:

Increase of biomass >16 after 72 hours; mean coefficient of variation for section-by-section specific growth rate <35%; coefficient of variation for average specific growth rate <7%.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, based on loading rates (OECD TG 201, EU C.3):
EL50: >100 mg/L, NOELR: ≥ 100 mg/L

Additional information

EL50  for freshwater algae >100 mg/L
NOELR for freshwater algae >100 mg/L

The results indicate that the test material is not inhibitory to growth of algae up to the limits of its water solubility (<1 mg/L) and a NOEC and thus a PNEC cannot be determined.