Fatty acids, tall-oil, compds. with oleylamine

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

Deviations:

yes

Remarks:

(on Days 0 and 1 of the test, the light intensity exceeded the mean by more than 15% (max 19%). This deviation did not adversely impact the results of this study.)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

yes

Remarks:

(on Days 0 and 1 of the test, the light intensity exceeded the mean by more than 15% (max 19%). This deviation did not adversely impact the results of this study.)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the test, two samples (50 mL) were taken from freshly prepared uninoculated control and test media. After 72 hours, two samples (50 mL) of control and test media were taken from uninoculated replicate flasks. Samples were stored in a freezer prior to analysis.

Vehicle:

no

Details on test solutions:

REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
4.77 mg/5 litres, 15.3 mg/5 litres,9.77 mg/1 litre, 31.3 mg/1 litre and 100 mg/1 litre was dispersed in dilution medium (algal nutrient medium as recommended in the guidelines) in glass vessels. The contents of each vessel were stirred for approximately 24 hours and then left to stand for approximately 24 hours. Aliquots were then removed from the mid-vessel region to provide the Water Accommodated Fractions (WAF) at nominal loading rates of 0.954, 3.05, 9.77, 31.3 and 100 mg/L. The WAF at nominal loading rate 3.05 mg/L was diluted with OECD medium to provide a nominal test concentration of 0.298 mg/L.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 25°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: 6 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:

25 mg/L as CaCO3

Test temperature:

22.5-24 °C

pH:

test start (0 h): 7.5-7.7
test end (72 h): 7.9-9.6

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: 0.0415, 0.0235, after 72 hours: 0.658, 0.316, 0.403, 0.116, 0.410 and 0.202 mg C/L (control mean: 0.726 mg C/L)
arithmetric mean: 0.350, 0.170, not applicable, not applicable, 0.252, 0.173

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: 1x10⁴ cells/mL
- Control end cells density: 121 x10⁴ 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: filtered, dechlorinated tap water, softened and treated by reverse osmosis before microfiltration and purification (resistivity 18 Megohm/cm)
- Total organic carbon: 0.4 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: nominally 5790 to 6112 lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. Light intensity (four corner positions and in a central position of the random block design) within the test area were determined each day. 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.
On Days 0 and 1, the light intensity exceeded the mean by 16-19%. As 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 24, 48 and 72 hours
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: nominal loading rates of 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: inhibition of algal growth after 72 hours was 100% and 74% at 100 and 10 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 4.5-8 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits: 4-8 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% condidence limits: 4-8 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3 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

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 4-7 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits: 3-6 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 3-6 mg/L; 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: minor stimulation was observed (see Table 2). This had no influemce on calculation of EC50 values.

At the start of the test, the test media (as WAF) at all nominal loading rates were colourless.

Results with reference substance (positive control):

- 72-hour EbC50: 0.541 mg/L (typical range in this laboratory: 0.3 to 1 mg/L)

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.

Table 1 Cell densities

Nominal loading rates (mg/L)	Replicate number	Cell densities (cells/mL)
		24 hours	48 hours	72 hours
Control	R1	63000	260067	1189500
	R2	58600	243100	1231633
	R3	54800	252167	1214800
	R4	59167	279067	1168433
	R5	56267	273833	1267533
	R6	56567	265000	1177467
	Mean	58067	262206	1208228
0.298	R1	54567	254033	1228367
	R2	63700	272133	1192367
	R3	60867	277933	1423267
	Mean	59711	268033	1281333
0.954	R1	56667	256900	1182933
	R2	65133	292100	1298233
	R3	57433	291233	1369733
	Mean	59744	280078	1283633
3.05	R1	58733	289833	1273633
	R2	57133	281233	1252300
	R3	60833	296367	1310433
	Mean	58900	289144	1278789
9.77	R1	1633	7100	13700
	R2	-5667	2000	7233
	R3	-4333	5600	7567
	Mean	-2789	4900	9500
31.3	R1	-2100	8367	10300
	R2	-2333	8000	6733
	R3	-4567	9233	7967
	Mean	-3000	8533	8333
100	R1	-900	3800	6633
	R2	-4433	7067	6900
	R3	-2367	6567	6467
	Mean	-2567	5811	6667

R1-R6: replicate number

Note: the initial cell density was estimated to be 0.875 x 10⁴/mL

 

 

Table 2 Inhibition of growth

  

Parameter	Nominal loading rates (mg/L)	Sample size	Mean	Inhibition (%)	p-value
Area under curve to 72 hours	Control	6	21.6	0	-
	0.298	3	22.6	-4.9	>0.999
	0.954	3	23	-6.4	>0.999
	3.05	3	23.1	-7.0	>0.999
	9.77	3	-0.4	102	<0.001***
	31.3	3	-0.4	101.7	<0.001***
	100	3	-0.4	102.0	<0.001***
Growth rate to 72 hours	Control	6	0.067	0.0	-
	0.298	3	0.067	-1.2	>0.999
	0.954	3	0.067	-1.2	>0.999
	3.05	3	0.067	-1.2	>0.999
	9.77	3	-0.001	102.0	<0.001***
	31.3	3	-0.003	104.1	<0.001***
	100	3	-0.006	108.5	<0.001***
Growth rate 0 to 24 hours°	Control	6	0.073	0.0	-
	0.298	3	0.074	-1.5	>0.999
	0.954	3	0.074	-1.6	>0.999
	3.05	3	0.074	-0.8	>0.999
	9.77	3	-0.076	203.1	<0.001***
Growth rate 24 to 48 hours	Control	6	0.063	0.0	-
	0.298	3	0.063	0.3	>0.999
	0.954	3	0.064	-2.5	0.553
	3.05	3	0.066	-5.6	0.553
	9.77	3	0.061	2.5	0.553
	31.3	3	0.063	0.0	-
	100	3	0.063	0.3	>0.999
Growth rate 48 to 72 hours	Control	6	0.064	0.0	-
	0.298	3	0.065	-2.2	>0.999
	0.954	3	0.063	0.4	>0.999
	3.05	3	0.062	2.7	>0.999
	9.77	3	0.031	51.1	<0.001***
	31.3	3	-0.002	102.4	<0.001***
	100	3	0.007	88.7	<0.001***
Yield	Control	6	1198228	0.0	-
	0.298	3	1271334	-6.1	>0.999
	0.954	3	1273633	-6.3	>0.999
	3.05	3	1268789	-5.9	>0.999
	9.77	3	-500	100.0	<0.001***
	31.3	3	-1667	100.1	<0.001***
	100	3	-3333	100.3	<0.001***

p values are for the comparison with Control using Williams' test

*** p < 0.001

° growth rate values at nominal loading rates of 31.3 and 100 mg/L between 0 to 24 hours could not be calculated as all cell counts at 24 hours were below the background measurements.

 

Control cultures:

-cell concentration increased by a factor of 121;

-the mean coefficient of variation for daily growth rates ranged between 2.8 and 4.6%;

- the coefficient of variation for the average specific growth rates was 0.64% during the 72 hour exposure period.

Validity criteria fulfilled:

yes

Remarks:

The criteria of OECD Guideline 201 and EU Method C.3 for biomass and growth rates were fulfilled.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, based on loading rates (OECD 201, EU C.3):
EL50: 7.4 mg/L, NOELR: 3.05 mg/L, EC10: 6.2 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

7.4 mg/L

EC10 or NOEC for freshwater algae:

3.1 mg/L

Additional information

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