Reaction product aqueous Phosphoric Acid, 2-Butoxyethanol and 4,4'-Isopropylidenediphenol-Epichlorohydrin

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 (Freshwater Alga and Cyanobacteria, 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:

no

Details on sampling:

No sampling for analysis conducted.

Vehicle:

no

Details on test solutions:

Since the test item is a mixture, which is not well soluble, the test solutions were prepared, separately for each test concentration, through 96-h stirring followed by filtration. The respective amounts of a test item stock solution, prepared in solvent (ethanol), were added to empty glass vessels. So that the total amount of solvent is equal in all vessels (i.e. equal to that of the highest concentration), the corresponding additional amount of pure solvent was added as well.

After complete evaporation of the solvent, the sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) was added. These mixtures were moderately stirred for 96 h, followed by filtration. The resulting solutions were used as test solutions in the toxicity test: 100 mL of each test solution were added to the sterile test flasks and inoculated with an exponentially growing preculture of algae. In the blank controls, pure solvent (ethanol) in a quantity equal to the one used for the highest test concentration was added and evaporated before addition of the algal medium, followed by 96-h stirring and filtration.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Exponentially growing culture used as per OECD guidelines.

ACCLIMATION
- Acclimation period: Not specified
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: Not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not specified

Test temperature:

21 to 24°C (maintained at ± 2°C in a thermo-controlled room)

pH:

Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal loading rates: 0, 3.16, 10, 31.6, 100 and 316 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass flasks
- Type (delete if not applicable): open (capped with air permeable stoppers)
- Material, size, headspace, fill volume: 100 mL of test medium.
- Aeration: No
- Initial cells density: 0.5 to 0.85 μg/mL with respect to dry weight corresponding to about 2–5x10^3 cells/ml (i.e. OD680 of about 0.005 units; all test solutions contain the same initial biomass of test alga)
- Control end cells density: Not specified
- No. of organisms per vessel: Starting density as above.
- No. of vessels per concentration (replicates): Three replicates
- No. of vessels per control (replicates): Six replicates
- No. of vessels per vehicle control (replicates): NA

GROWTH MEDIUM
- Standard medium used: yes (as per OECD guidelines)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not specified

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes for test medium ahead of testing
- Photoperiod: Continious illumination
- Light intensity and quality: 2933 to 2948 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell density every 24 hours to determine growth rate, biomass and yield at 72 hours.
- Determination of cell concentrations: spectrophotometer at 680 nm wavelength

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Standard as per guidelines.
- Range finding study: Yes
- Test concentrations: 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Some inhibition of growth rate was observed at 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 316 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Only of statistical significance and no biological relevance

Details on results:

In an initial definitive test, the results were inconsistent with the non-GLP range finding test and since the dose-response relationship seemed to be non-existent and non-monotonic, a second definitive test was performed. This data being more consistent, only the data of the 2nd definitive test was reported in full. The validity criteria with regards to controls were all met.

Results with reference substance (positive control):

The sensitivity of the currently used algae culture is in line with the level of sensitivity reported in guideline ISO 8692 “Water quality - Fresh water algal growth inhibition test with unicellular green algae, Third edition, 2012-02-15”, where a ring test with potassium dichromate yielded an ErC50 of 0.84 mg/l (SD = 0.12).

Reported statistics and error estimates:

The statistical significance of differences between effects observed for control and test item treatments was determined with a Welsh-t-Test after Bonferroni-Holm correction using the ToxRat Standard Version 3.2.1.

Average growth rate between 0 and 72 hours with inhibition values.

Loading rate (mg/L)	Replicate	Average growth rate	Average growth rate mean values	Coefficient of variation (%)	Inhibition (%)	Inhibition mean value (%)
0	A	1.461	1.41	4.3	-3.5	0
	B	1.471			-4.2
	C	1.470			-4.1
	D	1.367			3.2
	E	1.355			4.0
	F	1.346			4.7
3.16	A	1.363	1.36	0.4	3.4	3.9
	B	1.353			4.1
	C	1.354			4.0
10	A	1.185	1.20	1.1	16	15
	B	1.210			14.3
	C	1.204			14.7
31.6	A	1.188	1.18	0.6	15.8	16.2
	B	1.174			16.8
	C	1.185			16
100	A	1.043	1.08	3.4	26.1	23.1
	B	1.107			21.6
	C	1.105			21.7
316	A	1.126	1.13	2.6	20.2	20.2
	B	1.097			22.3
	C	1.155			18.2

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EL50 (growth rate) based on loading rates was higher than the maximum concentration tested (> 316 mg/L). The applicable NOELR was 3.16 mg/L based on statistics but biological relevence is questionable as less than 10% inhibition was observed compared to the controls.

Description of key information

The 72 hour EL50 (growth rate) based on loading rates was higher than the maximum concentration tested (> 316 mg/L). The applicable NOELR was 3.16 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

316 mg/L

EC10 or NOEC for freshwater algae:

3.16 mg/L