Alkenes, C15-18-branched and linear, maleated, hydrolyzed

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:

09 February 2009 to 02 June 2009

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

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and single loading rate of 100 mg/L
- Sampling method: Samples were taken at 0 and 72 hours for quantitative analysis
- Sample storage conditions before analysis: Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was prepared as a Water Accomodated Fraction (WAF)
- Differential loading: Single nominal loading rate at 100 mg/L

Due to the low aqueous solubility and complex nature of the test material for the purposes of the test, the test material was prepared as a Water Accomodated Fraction (WAF). 250 mg of the test material was added to the surface of 2.5 L of culture medium to give the 100 mg/L loading rate. After the addition of the test material, the culture medium was stirred by a magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture was allowed to stand for 1 hour. Visual inspection of the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was justifiable to remove the WAFs by filtering through a glass wool plug. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF was removed by mid-depth siphoning to give the 100 mg/L loading rate WAF. An aliquot of the WAF was inoculated with algal suspension to give the test concentration of 100 mg/L loading rate WAF.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 2726/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Not indicated
- Method of cultivation: the Master cultures were maintained in the laboratory by the periodic replenishment of culture medium


ACCLIMATION
- Acclimation period: The master cultures were maintained in the laboratory udner constant aeration and illumination at 21 ± 1 ºC.
- Culturing media and conditions (same as test or not): the culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post-exposure observation was carried out

Hardness:

Not measured.

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test.

pH:

The pH values of the control cultures were observed to increase from pH 7.3 at 0 hours to pH 7.6 ¿ 7.7 at 72 hours.

Dissolved oxygen:

Not measured.

Salinity:

Not measured.

Nominal and measured concentrations:

Table 1: Nominal and measured concentrations at 0 and 72 hours

Sample Nominal concentration LR WAF (mg/L) Concentration found (mg/L)
0 hours Control R1-R6 0.00922/ < LOQ
100 R1-R6 2.56
100 R4-R6 2.62
72 hours Control R1-R6 < LOQ
100 R1-R6 1.65
100 R4-R6 1.62

Details on test conditions:

250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L loading rate WAF treatment group. The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.25 x E+05 cells per mL. Inoculation of 2 litres of test medium with 18.8 mL of this algal suspension gave an initial nominal cell density of 4 x E+03 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 ¿ 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. Samples were taken at 0, 24, 47 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

From the data it was apparent that the growth rate, yield or biomass of Desmodesmus subspicatus were not affected by the presence of DCI-30.n over the 72 hour exposure period.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal ranges for this reference material. Exposure of Desmodesmus subspicatus to the reference material gave the following results:

ErC50 (0 - 72 h) : 0.52 mg/L, 95 % confidence limits 0.43 - 0.62 mg/L
EyC50 (0 - 72 h) : 0.29 mg/L, 95 % confidence limits 0.25 - 0.33 mg/L
EbC50 (0 - 72 h) : 0.30 mg/L, 95 % confidence limits 0.26 - 0.34 mg/L

NOEC (growth rate): 0.125 mg/L
NOEC (yield) : 0.125 mg/L
NOEC (biomass integral) : 0.0625 mg/L
LOEC (growth rate): 0.25 mg/L
LOEC (yield) : 0.25 mg/L
LOEC (biomass integral) : 0.125 mg/L

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loading rate WAF test group using the Student¿s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant decreases in growth rate (P ¿0.05) between the control and the 100 mg/L loading rate WAF test group and therefore the 'No observed effect loading rate' (NOELR) based on growth rate was 100 mg/L loading rate WAF.
There were no statistically significant decreases in yield between the control and 100 mg/L loading rate WAF (P ¿0.05) and, therefore the 'No observed effect loading rate' (NOEL) based on yield was 100 mg/L loading rate WAF.
There were no statistically significant decreases in biomass (P ¿0.05) integral between the control and 100 mg/L loading rate WAF test group and, therefore the 'No observed effect loading rate' (NOEL) based on yield was 100 mg/L loading rate WAF.

Chemical analyses of the test preparations at 0 hours showed a measured test concentration of 2.6 mg/L and was obtained from the 100 mg/L loading rate WAF. A decline in measured concentrations was observed at 72 hours in the range of 1.6 to 1.7 mg/L. This decline was considered to be due to adsorption of the test material to the algal cells present as the preliminary stability analyses conducted indicated that the test material was stable over the test duration.Whilst the preliminary recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not preclude long-term adsorption over the 72 -hour test period. Adsorption was not a factor in the preliminary stability analyses concucted as no algal cells were present.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, the results are based on nominal loading rates only.

Table 2: Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.063	0.054	0.081
	R2	0.070	0.046	0.082
	R3	0.067	0.042	0.086
	R4	0.070	0.042	0.085
	R5	0.071	0.050	0.073
	R6	0.066	0.054	0.075
	Mean	0.068	0.048	0.080

R₁- R₆= Replicates 1 to 6

Table 3: Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)		Biomass Integral (cells/mL/hour)
		0 ¿ 72 h	% Inhibition	0 ¿ 72 h	% Inhibition*	0 ¿ 72 h	% Inhibition
Control	R1	0.066		4.75E+05		7.69E+06
	R2	0.067		4.78E+05		7.78E+06
	R3	0.066		4.45E+05		7.12E+06
	R4	0.066	-	4.66E+05	-	7.50E+06	-
	R5	0.065		4.33E+05		7.42E+06
	R6	0.065		4.40E+05		7.39E+06
	Mean	0.066		4.56E+05		7.48E+06
	SD	0.001		1.94E+04		2.37E+05
100	R1	0.067	[2]	4.94E+05		8.11E+06	[8]
	R2	0.067	[2]	4.99E+05		8.16E+06	[9]
	R3	0.067	[2]	4.90E+05		7.92E+06	[6]
	R4	0.067	[2]	4.86E+05		7.78E+06	[4]
	R5	0.067	[2]	4.88E+05		8.09E+06	[8]
	R6	0.067	[2]	4.94E+05		8.25E+06	[10]
	Mean	0.067	[2]	4.92E+05	[8]	8.05E+06	[8]
	SD	0.000		4.57E+03		1.71E+05

*In accordance with the OECD test guideline only the mean value for yield is calculated

R₁¿ R₆= Replicates 1 to 6. SD= Standard Deviation

Table 4: Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate (mg/L)		pH	Cell Densities*(cells per mL)				pH
		0 h	0 h	24 h	47 h	72 h	72 h
Control	R1	7.3	4.36E+03	1.80E+04	6.31E+04	4.80E+05	7.7
	R2	7.3	4.98E+03	2.14E+04	6.16E+04	4.83E+05	7.7
	R3	7.3	6.50E+03	1.97E+04	5.20E+04	4.51E+05	7.7
	R4	7.3	4.78E+03	2.14E+04	5.65E+04	4.70E+05	7.6
	R5	7.3	4.20E+03	2.22E+04	6.98E+04	4.37E+05	7.6
	R6	7.3	4.08E+03	1.96E+04	6.74E+04	4.44E+05	7.6
	Mean		4.82E+03	2.04E+04	6.17E+04	4.61E+05
100	R1	6.2	5.18E+03	2.29E+04	6.57E+04	4.99E+05	7.1
	R2	6.2	4.58E+03	2.39E+04	6.45E+04	5.04E+05	7.0
	R3	6.2	5.24E+03	1.97E+04	6.27E+04	4.96E+05	7.0
	R4	6.2	4.78E+03	1.91E+04	5.96E+04	4.91E+05	7.0
	R5	6.2	4.40E+03	1.94E+04	7.14E+04	4.93E+05	7.0
	R6	6.2	5.18E+03	2.19E+04	7.21E+04	4.99E+05	7.0
	Mean		4.89E+03	2.12E+04	6.60E+04	4.97E+05

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6.

Table 5: Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.063	0.054	0.081
	R2	0.070	0.046	0.082
	R3	0.067	0.042	0.086
	R4	0.070	0.042	0.085
	R5	0.071	0.050	0.073
	R6	0.066	0.054	0.075
	Mean	0.068	0.048	0.080

R₁- R₆= Replicates 1 to 6.

Table 6: Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)		Biomass Integral (cells/mL/hour)
		0 ¿ 72 h	% Inhibition	0 ¿ 72 h	% Inhibition*	0 ¿ 72 h	% Inhibition
Control	R1	0.066		4.75E+05		7.69E+06
	R2	0.067		4.78E+05		7.78E+06
	R3	0.066		4.45E+05		7.12E+06
	R4	0.066	-	4.66E+05	-	7.50E+06	-
	R5	0.065		4.33E+05		7.42E+06
	R6	0.065		4.40E+05		7.39E+06
	Mean	0.066		4.56E+05		7.48E+06
	SD	0.001		1.94E+04		2.37E+05
100	R1	0.067	[2]	4.94E+05		8.11E+06	[8]
	R2	0.067	[2]	4.99E+05		8.16E+06	[9]
	R3	0.067	[2]	4.90E+05		7.92E+06	[6]
	R4	0.067	[2]	4.86E+05		7.78E+06	[4]
	R5	0.067	[2]	4.88E+05		8.09E+06	[8]
	R6	0.067	[2]	4.94E+05		8.25E+06	[10]
	Mean	0.067	[2]	4.92E+05	[8]	8.05E+06	[8]
	SD	0.000		4.57E+03		1.71E+05

*In accordance with the OECD test guideline only the mean value for yield is calculated

R₁¿ R₆= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Desmodesmus subspicatus to Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes gave EL50 values (Effective loading rate)based on nominal loading rates of greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It is assumed that the Effective Loading rate (EL) is equivalent to the Effective Concentration (EC).

According to Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes does not warrant classifiaction.

Executive summary:

A test was carried out to determine the toxicity of Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes to Desmodesmus subspicatus. Exposure of Desmodesmus subspicatus to Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes gave EL₅₀ values based on nominal loading rates of greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It is assumed that the Effective Loading rate (EL) is equivalent to the Effective Concentration (EC).

According to Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, Hydrolysed reaction products of furan-2,5-dione and C15-C18-(linear and branched)-alkenes does not warrant classifiaction.

Description of key information

An algal growth inhibiton test was carried out according to OECD Guideline 201 and EU MEthod C.3. Desmodesmus subspicatus was exposed to the test material for a period of 72 hours under a static system. The EL50 (72 h) was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

- Water Solubility: insoluble
- Pow: log10 Pow range: 6.78 to 9.23.
- Biodegradation in water: Not readily biodegradable: 22% degradation after 28 days (OECD 301B).
- Stability under the test conditions: The test material was shown to be unstable in the test medium in light and dark conditions.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

A study was carried out to determine the toxicity of Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes to Desmodesmus subspicatus. In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants.

The test organisms were exposed to the test material over a period of 72 hours in fresh water under static conditions.

Exposure ofDesmodesmus subspicatus to Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes gave EL₅₀values based on nominal loading rates of greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It is assumed that the Effective Loading rate (EL) is equivalent to the Effective Concentration (EC).

According to Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, Hydrolysed reaction products of furan-2,5-dione and C15-18-(linear and branched)-alkenes does not warrant classifiaction.