NO. 701 BASE

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:

28 February 2011 - 18 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Substance type: Yellow-brown powder
- Lot/batch No.: 00120R

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the final test samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter was retained for possible analysis of the residue.

Frequency at t=0 h, t=24 h and t=72 h
Volume 2 ml
Storage Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. As in the test group prepared at a loading rate of 32 mg/l one replicate had darker colour than the remaining replicates a separate sample was taken from this replicate. Two remaining replicates were pooled.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of the substance tested was a yellow-brown powder. The test substance was not completely soluble in test medium at the loading rates initially prepared.

Preparation of the test solutions started with individually prepared loading rates of 1.0, 3.2, 10, 32 and 100 mg/l applying a 2-day period of magnetic stirring to achieve maximum dissolution in test medium. The obtained aqueous mixture was filtered through a 0.45 µm membrane filter (Whatman, rc 55) and the clear and colourless filtrates were used as test concentrations. The lowest test concentration was prepared by subsequent dilution of the filtrate prepared at the loading rate of 3.2 mg/l in test medium.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10^4 cells/ml.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1.
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation):4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/ml.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

-

Post exposure observation period:

none

Test conditions

Hardness:

0.24 mmol(24 mg CaCO3/L)

Test temperature:

The temperature of the test medium was 22.2°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.3 and 23.6°C.

pH:

8.0 -8.3

Dissolved oxygen:

not measured; vessels were continously shaken

Salinity:

not measured

Nominal and measured concentrations:

See table below in materials and methods.
At the start of the test, the actual test concentrations were 0.81 mg/l, 1.5 mg/l, 7.3 mg/l, 11 mg/l, and 23 mg/l at the individually prepared loading rates of 1.0, 3.2, 10, 32 and 100 mg/l, respectively. In the solution containing 10% of the filtrate prepared at a loading rate of 3.2 mg/l the initially measured concentration was 0.14 mg/l. All concentrations remained stable during the first 24 hours of exposure (91-101% of initial).
At the end of the test, only the lowest concentration had slightly decreased (72% of initial), while all higher concentrations remained stable (90-103%of initial).
The concentration measured in the highest test concentration incubated without algae showed similar trend as corresponding concentration with algae.
Based on these results, the Time Weight Average (TWA) concentrations were calculated.

Details on test conditions:

FINAL STUDY:
TEST SYSTEM
- Type: open
- Material, size, headspace, fill volume: 100 ml, normal headspace, 50 ml
- Aeration: no
- Initial cells density: 10000 cells/ml
- Control end cells density: 1192000 cells/ml (rapport: Mean cell densities (x 104 cells/ml) during the final test)
Replicates:
3 replicates of each test concentration,
6 replicates of the control,
1 replicate of each concentration without algae,
1 or 2 replicates of each test concentration for sampling purposes.

GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 76 to 88 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: control and 0.45 µm filtered solutions prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/l. In addition a ten-fold dilution of the filtrate prepared at a loading rate of 3.2 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: -

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: -

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI 95%: 0.47 – 2.1 mg/l

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI 95%: 0.12 – 0.49 mg/l

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Ci 95% : 12 – 54 mg/l

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Ci 95% : 0.90 – 3.5 mg/l

Details on results:

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Inhibition of yield increased with increasing concentration of the test substance from 0.77 mg/l upwards resulting in 91% inhibition at the highest concentration tested. Statistically significant inhibition of yield was found at the TWA concentrations of 0.77 mg/l and higher (Bonferroni t test, α = 0.05).

Results with reference substance (positive control):

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.4 mg/l with a 95% confidence interval ranging from 1.1 to 1.9 mg/l.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.56 mg/l with a 95% confidence interval ranging from 0.34 to 0.94 mg/l

Reported statistics and error estimates:

Growth rates were in the range of the controls at the lowest concentration tested, whereas the growth rate of algae exposed to higher concentrations were increasingly reduced. Reduction of growth rate decreased as exposure progressed at all concentrations.
Statistically significant reduction of growth rate was found at the TWA concentrations of 0.77 mg/l and higher (Bonferroni t test, α = 0.05).

Inhibition of yield increased with increasing concentration of test substance from 0.77 mg/l upwards resulting in 91% inhibition at the highest concentration tested. Statistically significant inhibition of yield was found at the TWA concentrations of 0.77 mg/l and higher (Bonferroni t test, α = 0.05).

Any other information on results incl. tables

TWA^1conc. test substance (mg/l)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction^2(%)	x10^4cells/ml	Inhibition^2(%)
control	0.06640		118.87
0.12	0.06708	-1.0	124.33	-4.6
0.77	0.06138	7.6	82.50	30.6
1.5	0.05662	14.7	58.33	50.9
7.3	0.04557	31.4	25.83	78.3
11	0.03909	41.1	16.25	86.3
23	0.03336	49.8	10.17	91.4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, the test substance reduced growth rate and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 0.77 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 25 mg/l with a 95% confidence interval ranging from 12 to 54 mg/l, based on TWA concentrations.

The EC50 for yield inhibition (EYC50: 0-72h) was 1.8 mg/l with a 95% confidence interval ranging from 0.90 to 3.5 mg/l, based on TWA concentrations.

The NOEC for growth rate reduction and yield inhibition was 0.12 mg/l.