Condensation products of tetramethylenediamine with hydrogenated fatty acids of castor-oil

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples from control and test media were taken from freshly prepared uninoculated media at test start. At test end (72 hours) samples were taken from uninoculated replicate flasks incubated under the test conditions.
Samples were analysed without prior storage.

Vehicle:

no

Details on test solutions:

REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
Aliquots of the test substance were dispersed in dilution medium. The bulk preparations were stirred for approximately 24 hours in the dark, then left to stand for approximately 24 hours in the dark. An aliquot (3 L) was then syphoned from a mid-vessel location of each preparation vessel, of which the first litre was discarded, to give 2 L aliquots of Water Accommodated Fractions (WAF) which were used as test media with nominal loading rates of 100, 32, 10, 3.2 and 1.0 mg/L. The lowest nominal loading rate of 0.32 mg/L was prepared by dilution of an aliquot of the WAF at 1.0 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 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 a further three days to provide an inoculum in the log phase of growth.

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:

21.7-22.8 °C

pH:

test start (0 h): 7.79-7.92
test end (72 h): 7.91-8.82

Nominal and measured concentrations:

nominal loading rates: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L as WAF
measured levels: The measured levels of TOC were below the limit of quantification of the analysis method after background correction for the control medium.

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: 150x10⁴ 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: 0.98 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 5648 to 5736 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell densities measured at 24, 48 and 72 hours
- Determination of cell concentrations: electronic particle counter. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts. Nominal loading rates 0, 0.32, 1.0 and 3.2 mg/L were corrected against un-inoculated dilution media. The nominal loading rates of 10, 32 and 100 mg/L were corrected against un-inoculated WAF at the same loading rate as the test flask, as the initial cell counts for these treatment groups were higher than the background cell counts, indicating that the WAF’s at these levels were effecting the cell counts.

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 82% and 71% at 100 and 10 mg/L; no inhibition occurred at 1.0 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

46.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 28-89 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits: 0.2-2.5 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits: 4.1-5.9 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence limits: 1.1-2.8 mg/L; results are expressed in terms of loading rates

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

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 were colourless.

Results with reference substance (positive control):

- 72-hour ErC50: 0.73 mg/L (typical range in this laboratory: 0.3 to 1 mg/L)

Reported statistics and error estimates:

For determination of the ECx values the approaches recommended in the OECD guideline 201 were used. 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	45583	237267	1392783
	R2	48150	263800	1538617
	R3	46783	242233	1444350
	R4	45517	239067	1512217
	R5	49617	295900	1592783
	R6	51117	266567	1549983
	Mean	47794	257472	1505122
0.320	R1	53317	288133	1637750
	R2	51350	280433	1578150
	R3	51483	282900	1525150
	Mean	52050	283822	1580350
1.00	R1	50517	280167	1333550
	R2	52083	259567	1293583
	R3	45017	225333	1188017
	Mean	49206	255022	1271717
3.20	R1	35417	186333	1036083
	R2	43783	198933	1119417
	R3	51950	245767	1247917
	Mean	43717	210344	1134472
10.0 A	R1	7700	40267	199100
	R2	7133	32100	152767
	R3	10267	48100	196100
	Mean	8367	40156	182656
32.0 A	R1	5133	17567	99200
	R2	7567	32667	126867
	R3	8733	39600	159733
	Mean	7144	29944	128600
100 A	R1	-3000 B	19833	145600
	R2	-8633 B	14133	137433
	R3	-8967 B	-633	103333
	Mean	-6867 B	11111	128789

R_1-R₆   Replicate number.

Note: Initial nominal cell density = 0.974 x 10⁴cells/mL (Equipment used: Coulter Z Series Particle Count and Size Analyser)

A: Nominal loading rates of 10, 32 and 100 mg/L were corrected against un-inoculated WAF at the same loading rate as the test flask

B: Results were below the background level for the un-inoculated WAF at this concentration, therefore the values reported were negative, and statistical analysis could not be performed for this data.

 

Table 2  Inhibition of growth

 

Parameter	Nominal loading rates (mg/L)	Sample size	Mean	Inhibition (%)	p-value
Growth rate to 72 hours	Control	6	0.0696	0.0
	0.32	3	0.0703	-1.0	1.000^W
	1.0	3	0.0673	3.4	0.082^W
	3.2	3	0.0657	5.7	0.005**^W
	10	3	0.0402	42.2	<0.001***^W
	32	3	0.0352	49.4	<0.001***^W
	100	3	0.0353	49.2	<0.001***^W
Growth rate 0 to 24 hours	Control	6	0.0651	0.0
	0.32	3	0.0687	-5.5	1.000^W
	1.0	3	0.0663	-1.8	1.000^W
	3.2	3	0.0610	6.4	0.444^W
	10	3	-0.0080	112.2	<0.001***^W
	32	3	-0.0150	123.1	<0.001***^W
	100 A	3	A	A	A
Growth rate 24 to 48 hours	Control	6	0.0701	0.0
	0.32	3	0.0707	-0.9	1.000^W
	1.0	3	0.0685	2.3	0.592^W
	3.2	3	0.0657	6.3	0.088^W
	10	3	0.0653	6.8	0.066^W
	32	3	0.0584	16.7	<0.001***^W
	100	3	-0.1105	257.6	<0.001***^W
Growth rate 48 to 72 hours	Control	6	0.0737	0.0
	0.32	3	0.0715	2.9	0.969^D
	1.0	3	0.0671	9.0	0.208^D
	3.2	3	0.0704	4.4	0.824^D
	10	3	0.0634	13.9	0.021*^D
	32	3	0.0623	15.5	0.010**^D
	100	3	0.0889	-20.7	0.003**^D
Yield to 72 hours	Control	6	1495122	0.0
	0.32	3	1570350	-5.0	1.000^W
	1.0	3	1261717	15.6	<0.001***^W
	3.2	3	1124472	24.8	<0.001***^W
	10	3	172656	88.5	<0.001***^W
	32	3	118600	92.1	<0.001***^W
	100	3	118789	92.1	<0.001***^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

A: Results were negative; therefore statistical analysis was not performed (see Table 1, footnote B).

 

Control cultures:

- cell concentration increased by a factor of 150;

- the mean coefficient of variation (CoV) for section by section daily growth rates was 6.7%;

- the coefficient of variation for the average specific growth rates was 0.987% 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: 46.5 mg/L, NOELR: 1.0 mg/L, EL10: 0.85 mg/L

Additional information

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.