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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:

16 - 19 January 2018

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)

Version / remarks:

23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test medium, test item and algae (three replicates)
- Blank control (Bn); containing test medium and algae (three replicates)
- Since the test item is soluble, the test solution was prepared by dissolving the test item in sterile-filtered algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware and stirred for 15 minutes followed by ultrasonication until dissolution (approx. 15 minutes). 100 mL of this stock solution was added to the sterile test flasks and inoculated with an exponentially growing preculture of algae.
Samples for the determination of the test concentrations were taken from the remaining test solution. For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Culture: 250 mL flask containing 100 mL of sterile OECD medium inoculated with cell material from an axenic slope culture
- Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- Control of sensitivity: twice per year, with potassium dichromate

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

Min: 22.4; Max: 24; Mean: 23.2

pH:

7.6 - 8.3

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

Determination of the test item concentrations were based on DOC-analytics. DOC analyses of the test media were conducted at the beginning of the test (in the respective solutions before inoculation with algae) and at the end of the test (after 72 h). These analyses confirmed that the concentrations were correctly dosed (initially measured concentrations were within 95-107% of nominal concentrations) and showed that the DOC concentrations decreased over the 72h test period and were not within 80-120% of the nominal concentrations (53-165% of the nominal concentrations).
The effective concentrations ErCx/EyCx were assessed based on the nominal concentrations as well as on the geometric mean of the measured concentrations of the test item over the whole 72 h test period.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL flasks, all-glass, with 100 mL of test medium, shaken (130 rpm)
- Test medium: Recommended OECD medium
- Preculture: Exponentially growing culture of Desmodesmus subspicatus
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity: the light intensity amounts to about 4000 lux which corresponds to about 55 µE•m-2•s-2
- Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
- 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
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: 0.5–0.85 µg/mL with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/L of Yoghurt powder was performed.
The DOC determinations indicate that the test item concentrations were correctly dosed. Conclusions about their stability cannot however be drawn, since this quantification method is not substance specific. Based on the results of this non-GLP range finding test the test concentrations for the definitive test were 100, 31.6, 10.0, 3.16 and 1.00 mg/L nominal concentration.

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). Aliquots of 5 mL were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.

Reference substance (positive control):

not required

Remarks:

Control of sensitivity: twice per year, with potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

267 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: value extrapolated

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: determined by William's Test

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

60.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: determined by William's Test

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

164 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: value extrapolated

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: determined by William's Test

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: determined by William's Test

Details on results:

With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 26% at 100 mg/L, 7% at 31.6 mg/L and 3% at 10.0 mg/L. No significant effects were observed at 3.16 mg/L.
Based on these effects and the nominal concentrations of the test item, the median effect concentration with respect to growth rate (ErC50/0–3 days) of Yoghurt powder to Desmodesmus subspicatus was calculated to be 267 mg/L (95% confidence limits: 201–428 mg/L).
The low effect concentration with respect to growth rate ErC10 was calculated to be 38.6 mg/L (95% confidence limits: 28.2–46.9 mg/L).
The no observed effect concentration (NOErC) with respect to growth rate was determined to be 3.16 mg/L.

With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 67% at 100 mg/L, 27% at 31.6 mg/L and 12% at 10.0 mg/L. No significant effects were observed at 3.16 mg/L.
Based on these effects and the nominal concentrations of the test item, the median effect concentration with respect to yield (EyC50/0–3 days) of Yoghurt powder to Desmodesmus subspicatus was calculated to be 60.4 mg/L (95% confidence limits: 52.4–70.7 mg/L).
The low effect concentration with respect to yield EyC10 was calculated to be 12.5 mg/L (95% confidence limits: 8.50–16.3 mg/L).
The no observed effect concentration (NOEyC) with respect to yield was determined to be 3.16 mg/L.

Reported statistics and error estimates:

Statistical analysis was performed with respect to the no observed effect concentrations. The statistical analysis was conducted with the software ToxRat® Standard Version 3.2.1.

Validity criteria fulfilled:

yes

Remarks:

In the control cultures: average specific growth rate of 1.41 (>0.92)/ coefficient of variation of average specific growth rates: 1.1% (<=7%)/ mean coefficient of variation for section-by-section specific growth rates 17.4% (<=35%).

Conclusions:

The toxicity of Yoghurt powder (EC no. 917-734-0) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The 72 h ErC50 value of Yoghurt powder (EC no. 917-734-0) on the green alga Desmodesmus subspicatus is 267 mg/L nominal concentration corresponding to 164 mg/L mean measured concentration. The 72 h ErC10 value is 38.6 mg/L nominal concentration corresponding to 30.7 mg/L mean measured concentration.

Executive summary:

The growth inhibitory effects of Yoghurt powder (EC no.917-734-0) to the green alga Desmodesmus subspicatus were investigated according to test guideline OECD 201, over a period of 72 h.

The test item Yoghurt powder is 100% pure and soluble in algal medium. Consequently, the test solutions were prepared by respective dilutions of a stock solution.

The nominal concentrations were 100, 31.6, 10.0, 3.16 and 1.00 mg/L.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

The concentrations of Yoghurt powder in the test media were measured by DOC analysis at the beginning and at the end of the test. These analyses confirmed that the concentrations were correctly dosed (initially measured concentrations were within 95-107% of nominal concentrations) and showed that the DOC concentrations decreased over the 72h test period and were not within 80-120% of the nominal concentrations (53-165% of the nominal concentrations).Conclusions about the stability of the test item during the exposure phase cannot be drawn, since this quantification method is not substance-specific. The effective concentrations E_rC_x/E_yC_xwere assessed based on the nominal concentrations as well as on the geometric mean of the measured concentrations of the test item over the whole 72 h test period.

With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 26% at 100 mg/L, 7% at 31.6 mg/L and 3% at 10.0 mg/L. No significant effects were observed at 3.16 mg/L.

With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 67% at 100 mg/L, 27% at 31.6 mg/L and 12% at 10.0 mg/L. No significant effects were observed at 3.16 mg/L.

The results of the growth rate (r) and yield (y) inhibition of Yoghurt powder to the green alga Desmodesmus subspicatus are summarized in the following table showing EC values based on the nominal concentrations as well as on the geometric mean of the measured concentrations:

Parameter, expressed as Nominal concentration (mg(L)	ErC10	ErC50	NOErC	EyC10	EyC50	NOEyC
Estimate	38.6	267*	3.16	12.5	60.4	3.16
Parameter, expressed as Measured concentration (mg/L)	ErC10	ErC50	NOErC	EyC10	EyC50	NOEyC
Estimate	30.7	164*	2.75	10.2	45.4	2.75

*values > 100 mg/L are extrapolated

The 72 h ErC50 value of Yoghurt powder (EC no. 917-734-0) on the green alga Desmodesmus subspicatus is 267 mg/L nominal concentration corresponding to 164 mg/L mean measured concentration. The 72 h ErC10 value is 38.6 mg/L nominal concentration corresponding to 30.7 mg/L mean measured concentration.

All validity criteria fulfilled.

Description of key information

The toxicity of Yoghurt powder (EC no. 917-734-0) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The 72 h ErC50 value of Yoghurt powder (EC no. 917-734-0) on the green alga Desmodesmus subspicatus is 267 mg/L nominal concentration corresponding to 164 mg/L mean measured concentration. The 72 h ErC10 value is 38.6 mg/L nominal concentration corresponding to 30.7 mg/L mean measured concentration.

Key value for chemical safety assessment

EC50 for freshwater algae:

267 mg/L

EC10 or NOEC for freshwater algae:

38.6 mg/L