[Name confidential or not available]

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 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(on Days 1 and 2 of the test, the light intensity exceeded the mean by more than ±15% (-15 to +18%). This deviation did not adversely impact the results of this study.)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

(on Days 1 and 2 of the test, the light intensity exceeded the mean by more than ±15% (-15 to +18%). This deviation did not adversely impact the results of this study.)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the start of the test, four samples (50 mL) were taken from freshly prepared uninoculated control and test media. After 72 hours, four samples (50 mL) of control and test media were taken from the pooled contents of uninoculated replicate flasks. Samples were stored in a freezer prior to analysis.

Vehicle:

no

Details on test solutions:

REPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAF)
200 mg test material was dispersed in 2 L dilution medium (algal nutrient medium as recommended in the guidelines) in a glass vessel. The contents of the vessel were stirred for approximately 24 hours in the dark and then left to stand for approximately 24 hours. An aliquot (1200 mL) was then removed mid vessel to provide the Water Accomodated Fraction (WAF).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Three days. Algal cells used for inoculation were in the log phase of growth.
- Method of cultivation: Liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for three days and used as inoculum for the test.

ACCLIMATION
- Acclimation period: 6 days (pre-culturing)
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

25 mg/L as CaCO3

Test temperature:

23 °C

pH:

test start (0 h): 7.8
test end (72 h): 8.8

Nominal and measured concentrations:

nominal loading rates: 100 mg/L as WAF
measured levels at test start: below background - 0.13 mg C/L (mean: 0.069 mg C/L)
measured levels at test end: the mean measured level of TOC in the test medium was below that found in the control sample; so it was not possible to quantify the level of carbon that was attributable to the test substance

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flasks (autoclaved) containing 100 mL of inoculated test medium and loosly plugged with foam bungs
- Aeration/gaseous exchange: orbital incubator, oscillating at nominal 130 cycles per minute
- Initial cells density: 1x10⁴ cells/mL
- Control end cells density: 134x10⁴ cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium according to guidelines, using filtered, dechlorinated tap water, softened and treated by reverse osmosis before microfiltration and purification (resistivity 18 Megohm/cm)
- Total organic carbon: 2.2 mg C/L
- Ca/Mg ratio: 1:1
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: nominally 5870 to 6854 lux provided by 6 x 30 W “cool white” 1 metre fluorescent tubes. Light intensity (four corner positions and in a central position of the random block design) within the test area were determined each day. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the test.
On Days 1 and 2, the light intensity ranged between -15 and +18% of the mean. As the validity criteria were met, this deviation to guideline was not thought to have affected either the validity or integrity of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : cell densities measured at 24, 48 and 72 hours
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: nominal loading rates of 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no inhibition after 72 h

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:

other: growth rate, yield and biomass

Remarks on result:

other: results are expressed in terms of loading rates

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: results are expressed in terms of loading rates

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no microscopic abnormalities
- Any stimulation of growth found in any treatment: no biologically significant stimulation of growth was observed (see Tables 1 and 2).

At the start of the test, the test media (as WAF) at all nominal loading rates were colourless dispersions.

Results with reference substance (positive control):

- 72-hour EbC50: 0.541 mg/L (typical range in this laboratory: 0.3 to 1 mg/L)

Reported statistics and error estimates:

Statistical analysis was perfomed using SAS 9.1 (SAS Institute 2002), using nominal loading rates. 95% confidence intervals were calculated using the likelihood ratio method. Williams’ test was used to compare each treated group with control unless there was evidence of a non-monotonic dose-response relationship, in which case Dunnett’s test was used.

Table 1: Cell densities

 

Nominal loading rates	Replicate	Cell densities (cells/mL)
(mg/L)	number	24 hours	48 hours	72 hours
Control	R1	14750	232733	1346183
	R2	20817	215967	1358917
	R3	22650	221100	1239883
	R4	20250	255100	1326950
	R5	17183	259667	1362817
	R6	16617	211767	1403450
	Mean	18711	232722	1339700
100	R1	12317	196933	1234717
	R2	22083	233433	1365883
	R3	15317	203533	1401917
	R4	15317	240233	1469450
	R5	16783	211433	1417117
	R6	18617	252033	1591583
	Mean	16739	222933	1413444

 

R1-R6: replicate number

Note: the initial cell density was estimated to be 1.405 x 10⁴/mL

 

 

Table 2  Inhibition of growth

 

Parameter	Nominal loading rates (mg/L)	Sample size	Mean	Inhibition (%)	p-value
Area under curve to 72 hours	Control	6	21.5	0.0	-
	100	6	22.1	-2.8	0.489
Growth rate to 72 hours	Control	6	0.068	0.0	-
	100	6	0.069	-1	0.208
Growth rate 0 to 24 hours	Control	6	0.026	0.0	-
	100	6	0.021	19	0.289
Growth rate 24 to 48 hours	Control	6	0.105	0.0	-
	100	6	0.108	-2.9	0.484
Growth rate 48 to 72 hours	Control	6	0.073	0.0	-
	100	6	0.077	-5.4	0.068
Yield	Control	6	1329506	0.0	-
	10	6	1373039	-3.3	0.264
pvalues are for the comparison with Control using thet-test

 

 

Control cultures:

-cell concentration increased by a factor of 134;

-the mean coefficient of variation for daily growth rates ranged between 6 and 26%;

- the coefficient of variation for the average specific growth rates was 0.9% during the 72 hour exposure period.

Validity criteria fulfilled:

yes

Remarks:

The criteria of OECD Guideline 201 and EU Method C.3 for biomass and growth rates were fulfilled.

Conclusions:

The results indicate that the test material is not toxic to freshwater algae up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.

Description of key information

effect values for average specific growth rates of Pseudokirchneriella subcapitata, 0-72 h, based on loading rates (OECD TG 201, EU C.3):
EL50: >100 mg/L, NOELR: ≥ 100 mg/L 

Additional information

EL50, EL10/NOELR for freshwater algae > water solubility

The results indicate that the test material is not toxic to freshwater algae up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.