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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance.

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification document provided in IUCLID section 13.

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2 286.77 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

growth rate

Remarks on result:

other: source, RA-A, CAS 91050-80-5, Emery, 2007, Skeletonema costatum, 72h

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

growth rate

Remarks on result:

other: source, RA-A, CAS 91050-80-5, Emery, 2007, Skeletonema costatum, 72h

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: source, RA-A, CAS 91052-28-7, BASF, 2013, Desmodesmus subspicatus, 72h

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: source, RA-A, CAS 91052-28-7, BASF, 2013, Desmodesmus subspicatus, 72h

Conclusions:

No effects were observed up to the limit of water solubility.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun - 08 Jun 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given (comparable to guidelines/standards)

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: ln order to determine an appropriate test preparation method, an assessment was made of its behaviour in seawater. 1000 mg of the test material was added to filtered seawater, and the resulting mixture was stirred for approximately 20 - 24 hours. The mixture was then left to settle for approximately 1h, and its behaviour assessed (SOP 402). lf, on addition to seawater, the material produced floating, settled or neutrally buoyant particles or films, it was classified as poorly soluble and exposures were carried out with water accommodated fractions (WAFs) of the test material. WAFs were prepared by the direct addition of the required nominal weights or volumes to seawater followed by gentle stirring for approximately 20
hours followed by a settling period of approximately one hour. After this settling period, the middle phase of the preparation is siphoned, avoiding incorporation of undissolved particles, if present.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): floating or settled particles at 1000 mg/L in sea water after 20 hours stirring

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Culture number: C5B2 A
- Source (laboratory, culture collection): stock laboratory cultures
- Method of cultivation: Pre-cultures in the exponential growth phase were prepared from stock laboratory cultures by inoculating nutrient medium (ISO culture medium) to a cell density of approximately 2 x 10^3 to 10^4 cells per millilitre. The pre-cultures were incubated at approximately 20 ± 2 °C under constant illumination for 3d ± 1d, and were used as the inoculum source for subsequent toxicity tests.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

19.4 - 19.9 °C

pH:

7.96 - 8.11 (ISO culture medium & test material stocks)
8.11 - 9.00 (test control)

Salinity:

36‰

Nominal and measured concentrations:

Definitive test: 1000, 1800, 3200, 5600, 10000 mg/L (nominal, WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed; loosely covered with aluminium foil caps
- Material, size, headspace, fill volume: 100-mL borosilicate glass conical flasks, filled with 80 mL of test medium seawater
- Initial cells density: 10,000 cells per mL
- No. of vessels per concentration (replicates): 2

GROWTH MEDIUM
- Standard medium used: ISO culture medium. ISO medium is prepared from natural seawater supplied by pump from Scapa Flow, Orkney. All seawater was UV sterilised and filtered to 0.2 µm. The filtered treated seawater was then enriched with nutrients and vitamins in accordance with ISO guidelines. The salinity of the enriched natural seawater at 0 h was 35‰ ± 4‰.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Clean natural seawater with a salinity of 35 ± 4‰ at 0h is used in the test.
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH: At pre 0h or 0h, the pH of the ISO culture medium was adjusted if required, by adding 1M HCI, or NaOH to give a pH of 8 ± 0.2. If, at 0h the pH of the test material stock(s) was outwith the pH range of 8 ± 0.2 then the pH was returned to within these Iimits by adjustment with either 1M HCI or NaOH as was appropriate. lf the pH requires adjustment, a stirring period was required to ensure the pH remained constant.
- Photoperiod: Continuous cool white illumination. The illumination consists of four 40W tubes mounted at a distance of approximately 40 cm directly above the test area
- Light intensity and quality: 7590-9230 lux
- Salinity (for marine algae): 36‰

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Haemocytometer

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 4 controls, 1, 10, 100, 1000 mg/L (nominal, WAF)
- Results used to determine the conditions for the definitive study: provisional 72 h EC50: >1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorephenol

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

< 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

2 186.3 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1992.97 - 2364.76

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2 286.77 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 2235.33 - 2334.32

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test mat. WAF

Basis for effect:

growth rate

Results with reference substance (positive control):

3,5 Dichlorophenol EC50(72h): 2.70 mg/L (95% CL: 2.63 - 2.79 mg/L)

Reported statistics and error estimates:

The EC50 for each time interval and the 72h EC90 and NOEC values were calculated using an appropriate statistical method from the ToxCalc Version 5 software.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Aug - 24 Aug 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 23 on Aquatic Toxicity of Difficult Substances and Mixtures, OECD 2000

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance is a mixture that is poorly soluble in water. Therefore the test solutions were prepared following general guidance provided in OECD 23 in order to achieve a water accommodated fraction (WAF) of the test substance. Generally, each test solution was prepared separately (differential loading) by directly adding test substance to test medium. A defined volume of test media was placed into a beaker (2L nominal volume) and a glass sampling tube was suspended in the beaker with the top of the tube sufficiently above the surface of the test medium to avoid overflow from the surrounding surface layer. The test substance was pipetted carefully with a positive displacement pipette on the water surface outside of the glass tubes. The beakers were covered with Parafilm and shaken gently (approx. 50 RPM, to avoid emulsion formation) for about one day. After shaking, the required volume of aqueous fraction was drawn off for testing via the glass sampling tube. The exposure was started after separation of the undissolved material by adding inoculum culture to the WAF at a ratio of 1:100.
Undissolved test substance was visible outside the glass sampling tube at the water surface in all test treatments. Inside of the glass sampling tube no undissolved test substance was observed. All stock test solutions appeared colorless and clear.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: SAG 86.81
- Source (laboratory, culture collection): The algae were obtained at regular intervals from SAG (Collection of algal cultures in Göttingen) and were kept in liquid culture in the Laboratory of Ecotoxicology of the Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany
- Age of inoculum (at test initiation): A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).
- Inoculum culture: An inoculum culture in exponential growth phase was prepared with an aliquot of the stock algal culture added to sterile test media to provide an initial cell density of 0.5 x 10E04 cells/mL. The inoculum culture is incubated under test conditions for 4 days prior to test initiation. The increase in biomass is verified to ensure that growth is within the normal range and algal cells are examined microscopically for normal morphology prior to use for test inoculation. Inoculum culture cell density (4 days growth): 184x 10E04 cells/mL (368 fold increase).
Inoculum culture morphology: normal and healthy

ACCLIMATION
- Culturing media and conditions: Same as in the final test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.9 - 23.2 °C

pH:

7.5 - 8.0

Nominal and measured concentrations:

Nominal: control, 3.2, 10, 32, 100, and 320 mg/L (loading rate, WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge caps and filled with 100 mL test solution
- Test chamber: Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet
- Initial cells density: 0.5 x 10E04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes. The test medium was prepared according to OECD Guideline 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard medium according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: permanent
- Light intensity and quality: Artificial light, type universal white (OSRAM L 25); Average 7115 lux (within ± 15% variability) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: After the end of the exposure the control replicates were mixed and serially diluted by factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic
count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density
- Chlorophyll measurement: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wave length of 430 nm)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Results used to determine the conditions for the definitive study: The test concentrations were selected on the basis of a range finding test (experimental conduct in accordance with GLP but without a GLP Status). The results of the 72 hour range finding test were (as nominal concentrations): EC50 >100 mg/L for both fluorescence and growth rate. Pretest results show slight growth inhibition at 100 and 320 mg/L. Therefore this test was performed using a broad concentration range up to 320 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: not determined

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: not determined

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

22.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 9.41 - 54.1

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

54.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 31.0 - 96.4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

296 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 176 - 500

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

172 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 93.8 - 314

Details on results:

- Inhibition of growth rate was observed at nominal loading rates of 100 and 320 mg/L.
- All test solutions were visibly clear and colorless after preparation. There was no visible undissolved test substance and no other remarkable observations through the end of exposure (72 h).

Results with reference substance (positive control):

EC50 (72 h, growth) = 0.90 mg/L

Table: Growth inhibition test

Test Group (loading rate) [mg/L]	Mean Cell Density (cells/mL) x 10E04 at 72 hours	Mean yield at 72 hours	Mean specific growth rate at 72 hours
0 (control)	42.4	42.1	1.63
3.2	42.0	41.6	1.61
10	39.1	38.8	1.58
32	40.9	40.6	1.60
100	26.2	25.9	1.47
320	22.1	21.8	1.43

Description of key information

EL50 (72 h) = 2286.77 mg/L (nominal, Skeletonema costatum); read-across

NOELR (72 h) = 1000 mg/L (nominal, Skeletonema costatum); read-across

ErL50 (72h) > 320 mg/L (nominal, Desmodesmus subspicatus); read-across

ErL10 = 172 mg/L (nominal, Desmodesmus subspicatus); read-across

NOELR = 32 mg/L (nominal, Desmodesmus subspicatus); read-across

Additional information

There is no study available in which the toxicity of Fatty acids, C16-18 (even numbered), esters with glycerol oligomers to algae was assessed. Therefore, read-across to the structurally and chemically related source substances Fatty acids, C16-18, tetraesters with 3,3'-oxybis[1,2-propanediol] (CAS 91050-80-5) and Glycerides,C14-18 and C16-18 unsaturated, mono-, di- and tri- (CAS No. 91052-28-7) was performed in accordance with Regulation (EC) No 1907/2006 Annex XI, section 1.5 in order to fulfill the data requirements for this substance. The source substances are characterized by comparable chemical structures and are therefore considered suitable representatives for the assessment of the toxicity of the target substance to algae. A detailed read-across justification in provided in IUCLID section 13.

 

One experimental study investigating the toxicity of the source substance Fatty acids, C16-18, tetraesters with 3,3'-oxybis[1,2-propanediol] (CAS 91050-80-5) to marine algae was conducted according to ISO 10253 under GLP conditions. Skeletonema costatum was exposed to five nominal concentrations between 1000 and 10000 mg/L, which were prepared as Water Accommodated Fraction (WAF), in a static test for 72 h. Effects on growth rate were observed at the four highest test concentrations resulting in an EL50 (72 h) = 2286.77 mg/L (nominal) and NOELR (72 h) = 1000 mg/L (nominal). Although the test solutions were prepared as Water Accommodated Fractions (WAF), floating or settled particles were observed at the lowest test concentration of 1000 mg/L after 20 hours stirring. Therefore, the effects on growth rate at the four highest test concentrations are probably caused by physical effects due to undissolved test material. It is thus not expected that the reported EL50 refers to a systemic toxic effect.Nevertheless, it can be concluded that Fatty acids, C16-18, tetraesters with 3,3'-oxybis[1,2-propanediol] show no effects up to a loading rate of 1000 mg/L (nominal).

One study evaluating the toxicity of the source substance Glycerides,C14-18 and C16-18 unsaturated, mono-, di- and tri- (CAS No. 91052-28-7) to algae was performed according to OECD Guideline 201, under GLP conditions. Desmodesmus subspicatus was exposed for 72 hours at test concentrations of 3.2, 10, 32, 100 and 320 mg/L (nominal, loading rate). Chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm) was the measured test parameter. Nevertheless, cell density was also determined in order to derive the linear correlation between both parameters. After 72 hours of exposure, effects on algal growth rate were observed at concentrations of 100 mg/L and 320 mg/L, leading to an ErL10 of 172 mg/L and a NOELR of 32 mg/L (nominal, loading rate). The ErL50 was determined to be > 320 mg/L (nominal, loading rate). The reported effects are well above the water solubility of the test source substance and therefore, they could be caused by physical interference or adsorption between algae and the test substance.Nevertheless, stock solutions were reported to be colourless and clear, and therefore, effects due to toxicity cannot be discarded.

Based on the available results from structurally related read-across substances (in accordance with Regulation (EC) No 1907/2006 Annex XI, 1.5) which are characterized by a similar ecotoxicological profile and comparable structure, it can be concluded that the target substance Fatty acids, C16-18 (even numbered), esters with glycerol oligomers is not expected to show toxicity to algae up to the limit of its water solubility (91.9 µg/L).