HPSA

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August - 12 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part C.3: Methods for the Determination of Ecotoxicity: Algal Inhibition Test; Official Journal of the European Union, No. L 142.

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD (2006), Test No. 201: Alga, Growth Inhibition Test, OECD Guidelines for the Testing of Chemicals, Section 2: Effects on Biotic Systems, No. 390, OECD Publishing.

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

International Standard ISO 8692. Water quality - Freshwater algal growth inhibition test with unicellular green algae. Third edition, 2004

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

This test was conducted utilizing modified exposure media, closed test system, and reduced inoculation density. An additional reference toxicant test was conducted with the same method modifications described in this study to determine any effect on algal response to toxic stress. The results of a reference toxicant test indicate that the algae are responding normally to toxicant stress under the modified test conditions and that these deviations from test guidelines do not affect the results of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

LC-MS

Details on sampling:

- Concentrations: 0.5, 5, 10, 20 and 40 mg/L
- Sampling method: Approx. 40 mg of test substance, accurately weighed (semi-micro balance) were dissolved in water in a 100 mL volumetric flask as described in the method. The flask was filled to the mark with water and the resulting solution was thoroughly mixed (= stock solution, 400 mg/L). Calibration solutions with nominal concentrations of 0.5, 5, 10, 20 and 40 mg/L HPSA were prepared.
For system suitability testing, a second calibration solution was prepared suing independent weigh-in (=control solution, nominal concentration: 10 mg/L HPSA).
Each calibration solution was injected in duplicate (at the beginning and end of the sequence).
For the determination of HPSA in test items, test item solutions were prepared to produce concentrations in the range of 5-20 mg/L by dilution with ultrapure water. All test items were subject to ultracentrifugation (3 min, 15000 rpm).

- Sample storage conditions before analysis: No storage indicated

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions were prepared separately for each test concentration.
For this the test substance was pipetted in and was made up with OECD medium to reach concentrations of 111% of the nominal concentration to compensate for the dilution by the later addition of inoculum. To reach the correct nominal concentrations at test start, inoculum culture was added to each 111% stock test solution at a ratio of 1:10.
The pH of the two highest test solutions were adjusted after the preparation.
All test solutions were visibly clear following preparation.
- Controls: Test water without test substance but treated in the same way as the test substance solution.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All of the 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).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- 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.
- Reason for selection of the test species: Recommended algae species proposed by the test guideline
- 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).
- Method of cultivation: 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).

ACCLIMATION
- Culturing media and conditions (same as test or not): 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 0.3 x 104 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) : 195 x 104 cells/mL (650 fold increase)
Inoculum culture morphology: normal and healthy

- Any deformed or abnormal cells observed: The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.5 – 22.5 °C

pH:

7.1-7.4 (start of test); 9.5-9.6 (after 72h)

Nominal and measured concentrations:

0 (control), 15.5, 48.3, 155, 483, 1546 mg/L as nominal concentrations based on test substance mass without correction for purity, corresponding to:
0 (control), 3.2, 10, 32, 100, 320 mg/L as nominal concentrations based on the dimer content (20.7%) of the test substance.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 mL) closed with glass plugs; test volume: approx. 300 mL (Erlenmeyer flasks were completely filled)
- Type (delete if not applicable): closed
- Initial cells density: The cell density of the inoculum culture in exponential growth phase is determined and then adjusted to 3 x 10^4 cells/mL with OECD media so that a 1:10 dilution of inoculum will result in an initial cell density of 0.3 x 10^4 cells/mL in the test replicates. The
initial inoculum biomass was reduced in this test due to the closed test vessels.
- No. of vessels per concentration (replicates): 3 replicates for each test substance concentration; 1 additional replicate per test group for initial concentration control analysis only.
- No. of vessels per control (replicates): 6 replicates for the Control; 1 additional uninoculated (without algae) replicate per test group for background fluorescence correction and to determine the influence of the test design on test substance stability.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test medium (OECD medium) was prepared according to OECD Guideline for Testing of
Chemicals, No. 201 Algal growth inhibition test, Mar 2006, Annex 3. To accommodate testing in a closed system (see Mayer 2000; ISO/DIS 14442, 2004) the concentration of NaHCO3 in the final modified OECD medium was increased to 300 mg/L and was adjusted to pH 7 by the addition of 1M HCL. The test medium was sterilized after preparation.

OTHER TEST CONDITIONS
- Sterile test conditions: An inoculum culture in exponential growth phase was prepared with an aliquot of the stock algal culture added to sterile test media.
- Adjustment of pH: The pH of the stock was adjusted to 7.1 with 1 molar NaOH before dilution and inoculation.
- Photoperiod: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination, the test vessels were rearranged daily.
- Light intensity and quality: Average 5089 lux (within ± 15% variability) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counted under a microscope (Neubauer-counting chamber)
- Effect calculated parameters: Exponentially growing algae are cultured under defined conditions for several generations in the presence of the test substance, and cellular biomass over the exposure period is determined in relation to an untreated control.

TEST CONCENTRATIONS
- Range finding study
According to the test guidelines, at least 5 concentrations in a geometric series with a separation factor of ≤3.2 should be used, preferably encompassing the range from 5 – 75% inhibition of algal growth.
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):
EfluorescenceC50 > 100 mg/L; ErC50 > 100 mg/L.
- Test concentrations: 0 (control), 15.5, 48.3, 155, 483, 1546 mg/L as nominal concentrations based on test substance mass without correction for purity, corresponding to: 0 (control), 3.2, 10, 32, 100, 320 mg/L as nominal concentrations based on the dimer content (20.7%) of the test substance.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

other: based on the dimer content (20.7%) of the test substance

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

other: based on the dimer content (20.7%) of the test substance

Basis for effect:

growth rate

Remarks:

and yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

other: based on the dimer content (20.7%) of the test substance

Remarks:

based on the dimer content (20.7%) of the test substance

Basis for effect:

growth rate

Remarks:

and yield

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: According to the test ISO guideline 8692 the EC50 values of the reference substance potassium dichromate should be in the range: ErC50 = 0.92 – 1.46 mg/L after 72 hours for Pseudokirchneriella subcapitata.

The ErC50 (72 h) of the control substance potassium dichromate was 0.96 mg/L (Date of the last control experiment: 17 Jun 2013 , project number: 60E0063/04E006)

These results indicate that the algae are responding normally to toxicant stress.

An additional reference toxicant test was conducted with the same method modifications described in this study (modified exposure media, closed test system, and reduced inoculation density) to determine any effect on algal response to toxic stress.

The ErC50 (72 h) of the control substance potassium dichromate was 1.08 mg/L (Date of experiment: 28 Jun 2013 , project number: 60E0063/04E007)

These results indicate that the algae are responding normally to toxicant stress under the modified test conditions and that these deviations from test guidelines do not affect the results of this study.

Reported statistics and error estimates:

The commercial software "TOXRAT Professional 2.10” (ToxRat Solutions GmbH, Alsdorf, Germany) was used for the statistical evaluation of the data.

Validity criteria fulfilled:

yes

Conclusions:

The short-term toxicity to aquatic algae was determined in a well-documented guideline study compliant to GLP. Therefore, it can be considered as reliable without restrictions (Reliability 1). The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based upon analytically confirmed concentrations, the 72-hour EC50 for the parameters yield and growth rate were determined to be > 320 mg/L.

Executive summary:

A study was performed to assess the test item for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation No. 440/2008.

Test groups:

0 (control), 15.5, 48.3, 155, 483, 1546 mg/L as nominal concentrations based on test substance mass without correction for purity, corresponding to 0 (control), 3.2, 10, 32, 100, 320 mg/L as nominal concentrations based on the dimer content (20.7%) of the test substance.

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):

EfluorescenceC50 > 100 mg/L; ErC50 > 100 mg/L

Duration of exposure: 72 hours; closed test vessels

Test substance preparation:

The test solutions were prepared separately for each test concentration.

For this the test substance was pipetted in and was made up with OECD medium to reach concentrations of 111% of the nominal concentration to compensate for the dilution by the later addition of inoculum. To reach the correct nominal concentrations at test start, inoculum culture was added to each 111% stock test solution at a ratio of 1:10.

The pH of the two highest test solutions were adjusted after the preparation.

All test solutions were visibly clear following preparation.

Concentration control analysis:

All measured concentrations of the test substance in the test water deviated less than 20% from the nominal concentration.

Thus, the test substance was stable in solution under test conditions and nominal concentrations are an accurate representation of exposure levels maintained throughout the test period. Following recommendations in OECD 201 the results should be evaluated based on the nominal concentrations.

Test results:

The following effect concentrations (mg/L) were obtained after 72-h nominal concentrations based on the dimer content (20.7%) of the test substance:

Algal Yield [mg/L]		Algal Growth Rate[mg/L]
EyC10 confidence limits 95%:	> 320 n.d.	ErC10 confidence limits 95%:	> 320 n.d.
EyC20 confidence limits 95%:	> 320 n.d.	ErC20 confidence limits 95%:	> 320 n.d.
EyC50 confidence limits 95%:	> 320 n.d.	ErC50 confidence limits 95%:	> 320 n.d.

n.d.: not determined due to mathematical reasons or inappropriate data

Since the measured concentrations of the test substance in the test solutions were within ±20% of the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 April - 1 May 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Qualifier:

according to guideline

Guideline:

other: ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)"

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: German Water Hazard Classification Scheme (Bewertung Wassergefährdender Stoffe LTWS-Nr 10)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(The Department of Health of the Government of the United Kingdom)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material were used

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For the purpose of the definitive study the test material was prepared by a direct dispersion in sterile reverse osmosis water.
An amount of test material (256 mg) was dispersed in sterile reverse osmosis water and the volume adjusted to 200 ml to give a 1280 mg/I stock solution. Serial dilutions were made from this stock solution to produce 640, 320, 160, 80, 40, 20, 10, 5.0, 2.5, 1.25 and 0.625 mg/I stock solutions.
To an aliquot (80 ml) of each stock solution, nutrient stock solutions and bacterial suspension were added to give the required test concentration range of 0.50, 1.0, 2.0, 4.0, 8.0, 16, 32, 64, 128, 256, 512 and 1024 mg/I.
Following the recommendations of the German Water Hazard Classification Scheme and ISO 10712, three identical test concentration ranges were produced as above from three separate weighings of the test material.
Analysis of the concentration, homogeneity and stability of the test material in the test solutions were not appropriate to the Test Guideline.

Test organisms (species):

other: Pseudomonas putida strain NCIB 8248

Details on test organisms:

TEST ORGANISM
- Common name: Pseudomonas putida
- Strain: NCIB 8248
- Source : National Collection of Industrial Bacteria , Aberdeen , Scotland
- Method of cultivation: An aqueous suspension of Pseudomonas putida was produced by adding pre-culture medium (see at Any other information on materials and methods incl. tables) to a stock culture of the bacterium and gently shaking in order to wash the bacterial cells off the solid medium . The resultant suspension was dispersed into a sterile flask plugged with sterile non-absorbent cotton wool and incubated at 25°C.
After the initial incubation period of approximately 18 hours, the bacterial suspension was diluted using pre-culture medium to give a turbidity of approximately 100 Formazine Turbidity Units (FTU). An aliquot (50 ml) of the 100 FTU bacterial suspension was added to 450 ml of pre-culture medium and incubated at 25°C. After incubation at 25°C for approximately 6 hours the bacterial suspension had a turbidity of approximately 50 FTU.

ACCLIMATION
- Culturing media and conditions : same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Hardness:

No data

Test temperature:

25°C

pH:

no data

Dissolved oxygen:

no data

Nominal and measured concentrations:

Range-finding study :
Nominal test concentrations : 6.4 , 80 , 1000 and 10000 mg/l

Definitive study :
Nominal test concentrations : 0.5, 1.0, 2.0, 4.0, 8.0, 16, 32, 64, 128, 256, 512 and 1024 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: glass conical flasks
- Type : plugged with sterile non-absorbent cotton wool
- Material, size, fill volume: 250 ml glass conical flasks filled with 100 ml test solution
- Aeration: none
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 10

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no


EFFECT PARAMETERS MEASURED :
- absorbance values determined for each control and test culture (at 436nm)
The absorbance of each test and control culture at 436 nm was determined using a Jenway 6100 spectrophotometer. The spectrophotometer was zeroed using sterile reverse osmosis water.

Reference substance (positive control):

no

Duration:

16 h

Dose descriptor:

EC10

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

16 h

Dose descriptor:

EC50

Effect conc.:

480 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

16 h

Dose descriptor:

NOEC

Effect conc.:

128 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Other:
The following data show that the inoculum used in the control multiplied by a factor of 105 during the test in line with the ISO Guideline that states the multiplication must be at least by a factor of 60 after 16 hours .
Mean absorbance of control at 0 hours : 0,008
Mean absorbance of control at 16 hours : 0,839

Reported statistics and error estimates:

Statistical analysis of the absorbance values was carried out for the control and all test concentrations using a Students t-test. There were no statistically significant differences between the control and 0.50, 1.0, 2.0, 4.0, 8.0, 16, 32, 64 and 128 mg/I test concentrations (p≥0.05), however all other test concentrations were significantly different (p<0.001) and, therefore the "No Observed Effect Concentration" (NOEC) is given as 128 mg/I.

Table 1 : Mean absorbance values from the range-finding study

Nominal Concentration	Absorbance Value
(mg/l)	0 hours	16 hours
Control	0,013	1,413
6,4	-	1,402
80	-	0,815
1000	-	0,074
10000	-	0,220

- = not determined

Table 2 : Mean absorbance values from the definitive study

Nominal Concentration	Absorbance Value
(mg/l)	0 hours	16 hours
Control	0,008	0,839
0,50	-	0,859
1,0	-	0,870
2,0	-	0,840
4,0	-	0,855
8,0	-	0,839
16	-	0,855
32	-	0,820
64	-	0,815
128	-	0,839
256	-	0,693
512	-	0,014
1024	-	0,014

- = not determined

Table 3 : Inhibition of growth

Nominal Concentration	H Values (%)
(mg/l)
0,50	- 2
1,0	- 4
2,0	0
4,0	- 2
8,0	0
16	- 2
32	2
64	3
128	0
256	18
512	99
1024	99

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the ISO guideline were fulfilled.

Conclusions:

The study is regarded as a valid guideline study with certificated GLP compliance. The effect of the test item on the growth of Pseudomonas putida has been investigated and gave an EC10(16h) of 180 mg/l and an EC50(16h) of 480 mg/l . The EC10 of 180 mg/I (180 ppm = 1.8 x 10-4 kg/l) corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of 3.7.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the bacteria Pseudomonas putida.The method followed that described in the German Water Hazard Classification Scheme (Bewertung Wassergefährdender Stoffe LTWS - Nr 10) and ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)". Following a preliminary range-finding study, Pseudomonas putida was exposed to an aqueous dispersion of the test material at concentrations of 0.50, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, 128.0, 256.0, 512.0 and 1024.0 mg/I (three replicate flasks per concentration) for approximately 16 hours, at a temperature of 25°C.

Samples of the bacterial populations were removed after approximately 16 hours and absorbance values determined for each control and treatment group.

Exposure of Pseudomonas putida to the test material gave an EC10 of 180 mg/I and an EC50 of 480 mg/l. The EC10 of 180 mg/I (180 ppm = 1.8 x 10

-4 kg/l) corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of 3.7. Statistical analysis of the absorbance values was carried out for the control and all test concentrations using a Students t-test. There were no statistically significant differences between the control and 0.50, 1.0, 2.0, 4.0, 8.0, 16, 32, 64 and 128 mg/I test concentrations (p≥0.05), however all other test concentrations were significantly different (p<0.001) and, therefore the "No Observed Effect Concentration" (NOEC) is given as 128 mg/I.

Description of key information

Pseudokirchneriella subcapitata, OECD 201 "Alga, Growth Inhibition Test", EC50(72h) > 320 mg/L

Pseudomonas putida strain NCIB 8248, ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)", EC50(16h) = 480 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

320 mg/L

EC10 or NOEC for freshwater algae:

320 mg/L

Additional information

A study was performed to assess the test item for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation No. 440/2008.

Test groups:

0 (control), 15.5, 48.3, 155, 483, 1546 mg/L as nominal concentrations based on test substance mass without correction for purity, corresponding to 0 (control), 3.2, 10, 32, 100, 320 mg/L as nominal concentrations based on the dimer content (20.7%) of the test substance.

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):

EfluorescenceC50 > 100 mg/L; ErC50 > 100 mg/L

Duration of exposure: 72 hours; closed test vessels

Test substance preparation:

The test solutions were prepared separately for each test concentration.

For this the test substance was pipetted in and was made up with OECD medium to reach concentrations of 111% of the nominal concentration to compensate for the dilution by the later addition of inoculum. To reach the correct nominal concentrations at test start, inoculum culture was added to each 111% stock test solution at a ratio of 1:10.

The pH of the two highest test solutions were adjusted after the preparation.

All test solutions were visibly clear following preparation.

Concentration control analysis:

All measured concentrations of the test substance in the test water deviated less than 20% from the nominal concentration.

Thus, the test substance was stable in solution under test conditions and nominal concentrations are an accurate representation of exposure levels maintained throughout the test period. Following recommendations in OECD 201 the results should be evaluated based on the nominal concentrations.

Test results:

The following effect concentrations (mg/L) were obtained after 72-h nominal concentrations based on the dimer content (20.7%) of the test substance:

Algal Yield [mg/L]		Algal Growth Rate[mg/L]
EyC10 confidence limits 95%:	> 320 n.d.	ErC10 confidence limits 95%:	> 320 n.d.
EyC20 confidence limits 95%:	> 320 n.d.	ErC20 confidence limits 95%:	> 320 n.d.
EyC50 confidence limits 95%:	> 320 n.d.	ErC50 confidence limits 95%:	> 320 n.d.

n.d.: not determined due to mathematical reasons or inappropriate data

Since the measured concentrations of the test substance in the test solutions were within ±20% of the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration.

 

Another study was performed to assess the effect of the test material on the growth of the bacteria Pseudomonas putida.The method followed that described in the German Water Hazard Classification Scheme (Bewertung Wassergefährdender Stoffe LTWS - Nr 10) and ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)". The study is regarded as a valid guideline study with certificated GLP compliance. The validity criteria of the ISO guideline were fulfilled. Following a preliminary range-finding study, Pseudomonas putida was exposed to an aqueous dispersion of the test material at concentrations of 0.50, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, 128.0, 256.0, 512.0 and 1024.0 mg/I (three replicate flasks per concentration) for approximately 16 hours, at a temperature of 25°C. Samples of the bacterial populations were removed after approximately 16 hours and absorbance values determined for each control and treatment group.

Exposure of Pseudomonas putida to the test material gave an EC10 of 180 mg/I and an EC50 of 480 mg/l. The EC10 of 180 mg/I (180 ppm = 1.8 x 10

-4 kg/l) corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of 3.7. Statistical analysis of the absorbance values was carried out for the control and all test concentrations using a Students t-test. There were no statistically significant differences between the control and 0.50, 1.0, 2.0, 4.0, 8.0, 16, 32, 64 and 128 mg/I test concentrations (p≥0.05), however all other test concentrations were significantly different (p<0.001) and, therefore the "No Observed Effect Concentration" (NOEC) is given as 128 mg/I.