Acetic acid, esters with lanolin alcs.

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-05-10 to 2001-07-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study from supporting substance (structural analogue)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

24th July 2001

Test material

Test material information

Constituent 1

Reference substance name:

Lanolin Alcohols

IUPAC Name:

Lanolin Alcohols

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
DOC concentration (mg/L) : 2.5 (measured), 1.3 (calculated), 0.6 (calculated), 0.3 (calculated), 0.15 (calculated)

- Sampling method:
Not reported

- Sample storage conditions before analysis:
Not reported

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
A saturated solution was prepared with test medium to make stock solution. A 1000 mg/L dispersion was prepared and shaken for 24 h in the
dark. After the dispersion treatment the solution was membrane filtrated (SCHLEICHER & SCHÜLL, ME 24, REF.-No. 401 714, 0.2 µm).

Test concentrations (nominal.): 1:16, 1:8, 1:4, 1:2 and 1:1 dilution out of stock solution

- Controls: Test medium (without test item).

- Chemical name of vehicle :
water

- Concentration of vehicle in test medium:
Not applicable

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: Strain CHODAT SAG 86.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Gottingen. Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE/m2 . s for 24 h per day.
- Age of inoculum: A four day old preculture was used.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE/m2 . s for 24 h per day.

ACCLIMATION
- Acclimation period: Not recorded.
- Culturing media and conditions: Nutrient medium Z according to LOTTGE et al. (1994).
- Any deformed or abnormal cells observed: None recorded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded

Test temperature:

Nominally 23 ± 2 °C
Measured continously by a Hygro-thermographe (KLIMATHERM)

pH:

8.01 - 8.90
Measured by a pH-Meter, pH 191 (WTW)

Dissolved oxygen:

Not recorded.

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Concentrations (nominal): 1:16, 1:8, 1:4, 1:2 and 1:1 dilution out of stock solution
Measured: 0.15 mg/L
Calculated: 0.3, 0.6, 1.3 2.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 100ml
- Aeration: No aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: Incubation was performed in 500 mL Erlenmeyer flasks with test medium. For the start of the test the preculture was diluted with test medium to receive an initial cell concentration of approximately 1 x 10E4 cells/mL in the replicates. All algae were from the same source and have not been used in a previous study.

- Control end cells density: algal cell density was approximately 6.24 10E5 cells/ml
- No. of organisms per vessel: initial cell concentration of approximately 1 x 104 cells/mL
- No. of vessels per concentration (replicates): Three replicate flasks per concentration.
- No. of vessels per control (replicates): Six replicate flasks.

TEST MEDIUM
According to the guidelines.

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No adjustments recorded
- Photoperiod: 24 h/d light
- Light intensity and quality: Nominally 60 - 120 µE/m2 .s (according to the EEC Guideline)

EXPOSURE CONDITIONS:
A definitive test was performed with 5 concentration levels in a geometrical series with a factor 2 and 3 replicates each. A control with test medium (without test item, six replicates) was tested under the same conditions as the test group. A four day-old preculture incubated at study conditions was used for the main study. Chlorophyll-fluorescence was determined at the beginning, after 24, 48 and 72 h.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ECxvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

- Chlorophyll measurement:
Cell density was measured via Chlorophyll-a-fluorescence (excitation at 435 nm, emission at 685 nm). Each replicate was measured 6 times.The cell density was measured at the beginning of the test and every 24 h. The control group was maintained under identical conditions but not exposed to the test material.

VALIDATION:
The cell density had to increase at minimum 16-fold in the control replicates within 72 h.

The temperature during the test had to be in the range of 21-25 °C controlled at ± 2°C.

The pH-value of the control replicates should not normally deviate by more than 1.0 units during the test.

DEVIATIONS FROM THE GUIDELINE: According to the guideline a light intensity of 120 IJE/m2. s ± 20 % is required. For the study the light intensity amounted 60 120 µE/m2 .s for 24 h per day according to EEC guideline. Yet the increase of cell density 16-fold was guaranteed.

DEVIATIONS FROM THE STUDY PLAN: Fluorescent tubes for alga culture was changed. Identification data were completed according to the sponsor's
wish. Title of the study was corrected. DOC of the saturated solution was measured.

These deviations had no impact on quality and integrity of the study.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence limits not reported

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits not reported

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits not reported

Details on results:

Definitive Test
The growth inhibition effects of the test item are summarized in Table 2. All data are given based on the initially measured DOC.

Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities.

Water quality parameters of pH-value, measured at 0 and 72 h, and room temperature, measured continuously, met the guideline requirements.

Results with reference substance (positive control):

The acute toxicity of potassium dichromate to the unicellular freshwater green alga Scenedesmus subspicatus was determined over a period of 72 h from December 4 to 7, 2000.

The EC50 - values of the reference item potassium dichromate after 72 h met the validity criteria according to EEC Directive 92/69/EEC Method C.3 Annex 2 (prescribed ranges are 0.20 - 0.75 mg/L and 0.60 - 1.03 mg/L potassium dichromate for inhibition of biomass growth and raterelated
inhibition, respectively).

The results with the reference substance are shown in table 1.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Table. 1: EC₅₀Values of Biomass Inhibition and Rate-Related Inhibition of the Reference item based on nominal concentrations mg/L, (0-72 h)

	Inhibition of biomass growth[mg/L]
EbC50	0.48
95% confidence interval	0.42 - 0.55
	Rate-related inhibition [mg/l]
ErC50	0.85
95% confidence interval	0.69 - 1.04

Table 2: Results (72 hours)

Test Material (mg DOC/L) Initially Measured Concentration	Replicate	Inhibition of Biomass (%)	Biomass Integral	Inhibition of Biomass (%)	Growth Rate Inhibition (%)
2.5 Saturated Solution	1	450874	18.50	1.18	10.14
	2	450356	18.60	1.18	10.24
	3	472452	14.60	1.17	11.22
	Mean	(+)457895	17.24	(+)1.18	10.55
1.3 (1:2)	1	468949	15.24	1.27	3.77
	2	465034	15.95	1.22	6.92
	3	498764	9.85	1.20	9.08
	Mean	(+)477582	13.68	(+)1.23	6.74
0.6 (1:4)	1	369589	33.20	1.13	13.96
	2	439126	20.63	1.17	11.02
	3	337689	38.96	1.16	11.96
	Mean	(+)382134	30.93	(+)1.15	12.27
0.3 (1:8)	1	365777	33.89	1.11	15.53
	2	441938	20.12	1.19	9.67
	3	452024	18.30	1.21	7.72
	Mean	(+)419913	24.10	(+)1.17	10.75
0.15 (1:16)	1	519822	6.04	1.33	-1.38
	2	512552	7.36	1.35	-3.00
	3	540723	2.26	1.37	-4.20
	Mean	(-)524366	5.22	(-)1.35	-2.85
Control	1	512450		1.29
	2	540188		1.31
	3	543249		1.29
	4	555022		1.34
	5	577830		1.30
	6	590767		1.36
	Mean	553250		1.32

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the teat material caused only minor effects on the freshwater green alga Scenedesmus subspicatus when tested up to the saturated aqueous solution. No dose-effect-relationship was observed. The EC50-values for inhibition of biomass growth (EbC50 ) and specific growth rate (ErC50 ) after 72 h were> 2.5 mg DOC/L for both end points.

Executive summary:

The acute toxicity of the test material to the unicellular freshwater green algaScenedesmus subspicatuswas determined according to the principles of OECD- Guideline 201, from June 12 to 15, 2001.

The study was conducted under static conditions over a duration of 72 hours with an initial cell density of nominally 10^4 cells/ml. A saturated solution served as stock solution and further 4 dilution levels in a geometrical series with a dilution factor 2 were prepared from this solution as listed in the following table.Corresponding DOC concentrations are as follows:

Test item concentrations

Dilution of stock solution	DOC concentration
	[mg/L]
1:1	2.5 (measured)
1:2	1.3 (calculated)
1:4	0.6 (calculated)
1:8	0.3 (calculated)
1:16	0.15 (calculated)

3 replicates were tested for each concentration level and 6 replicates for control.

The saturated solution and all dilution levels were found to be clear throughout the test.

Microscopic evaluation of the cells at the start of the incubation period revealed no morphological abnormalities. At the end cells at concentration levels of 0.3 to 2.5 mg DOC/L were larger as in control.

Water quality parameters of pH-value, measured at 0 and 72 h, and room temperature, measured continuously, were determined to be within the acceptable limits.

The effects of the test material, based on the initially measured DOC concentrations are summarized in the following table.

NOEC and ECso Values of Biomass Inhibition and Rate-Related Inhibition based on the initially measured DOC [mg/L] of the saturated solution, (0-72 h) NOEC calculated using one way analysis of variance and DUNNETT'S test.

	Inhibition of biomass growth [mgDOC/L]
EbC50	> 2.5
95% Confidence interval	Confidence interval
NOEC	0.15
	Rate-related inhibition [mgDOC/L]
ErC50	> 2.5
95% Confidence interval	Confidence interval
NOEC	0.15

Conclusion

In this study the test material caused only minor effects on the freshwater green alga Scenedesmus subspicatus when tested up to the saturated aqueous solution. No dose-effect-relationship was observed. The EC50-values for inhibition of biomass growth (E_bC₅₀) and specific growth rate (E_rC₅₀) after 72 h were > 2.5 mg DOC/L for both end points.