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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July to 25 July 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted by a recognised facility in compliance with GLP to a recent regulatory test guideline.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
see below
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The characterization of the test substance, the stability of the test substance and periodic analyses of well water for potential contaminants under conditions of storage at the test facility were not determined in compliance with Good Laboratory Practice.
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Every concentration and control group.
- Sampling method: Samples of the test solutions were collected at approximately 0 and 96 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches whereas at test termination samples were collected from the pooled replicate from the pooled replicates from each treatment and control group.
- Sample storage conditions before analysis: All samples were collected in glass vials and were analyzed immediately without storage.
Vehicle:
no
Details on test solutions:
A primary stock solution was prepared by dissolving Reofos 65 in freshwater algal medium at a nominal concentration of 2.5 mg/L. The stock was mixed in a 4 L Pyrex aspirator bottle with tabulation using a Teflon coated magnetic stir bar and a magnetic stir plate. The stock was allowed to mix overnight followed by a 1 hour setting period. The primary stock was proportionally diluted with freshwater algal medium to prepare the four additional test solutions at nominal concentrations of 0.16, 0.31, 0.63 and 1.3 mg/L. Test concentrations were not corrected for percent active ingredient in test substance. All test solutions appeared clear and colorless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Freswater green alga
- Strain: Selenastrum capricomutum Printz (UTCC 37)
- Source (laboratory, culture collection): The original source of the algae was the University of Toronto Culture Collection. The algae had been maintained in culture medium at Wildlife International, Ltd.
- Age of inoculum (at test initiation): no data.

ACCLIMATION
- Acclimation period: no data.
- Culturing media and conditions (same as test or not): no data.
- Any deformed or abnormal cells observed: no data.

IN LIFE DATES: 15 - 19 July 2005.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
All observations made during exposure period.
Hardness:
No data.
Test temperature:
24 ± 2°C

pH:
Initial pH: 7.9. Final pH values were in the range 8.3-8.9.

Dissolved oxygen:
No data.
Salinity:
Not applicable.
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations: 0.16, 0.31, 0.63, 1.3 and 2.5 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flaslcs
- Material, size, headspace, fill volume: 250-mL plugged with foam stoppers
- Aeration: no data.
- Initial cells density: 10,000 cells/mL
- Control end cells density: no data.
- No. of organisms per vessel: 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Stock nutrient solutions
were prepared by adding reagent-grade chemicals to purified Wildlife International, Ltd. well water. The
test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to purified
well water (NANO pure water). The pH of the medium was adjusted to 8.1 using 10% HCl.
The medium was sterilized by filtration (0.22 µm) prior to use.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: 24 hours per day of cool-white fluorescent lighting
- Light intensity and quality: 4300 ± 10 % lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Temperature: The temperature of a container of water adjacent to the test chambers in the environmental chamber was recorded twice daily during the test using a liquid-in-glass thermometer. In addition, temperature in the environmental chamber was measured continuously using a min/max. thermometer.
- Light intensity: Light intensity was measured at five locations surrounding the test flasks at test solution level at test initiation using a SPER Scientific 840006C light meter.
- pH: The pH of the medium in each treatment and control group was measured at test initiation and test termination using a Thermo Orion Model 720 Aplus pH meter. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and the control group. At test termination, pH was measured in pooled samples of test solution collected from each of the replicates of each treatment and control group.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Various
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell density
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: area under growth curve
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell density
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: area under growth curve
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell density
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: area under growth curve
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: cell density
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: area under growth curve
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Nominal concentrations selected for use in this study were 0.16, 0.31,0.63, 1.3 and 2.5 mg/L. Samples collected on Day 0 had recoveries of 50,29,32, 19 and 13% of nominal concentrations, respectively. Samples collected on Day 4 had recoveries of
Temperatures ranged from 22.7 to 24.5 deg C and were within the 24 +/- 2°C range established for the test. The minimum and maximum temperature measured continuously throughout the study were22.8 and 24.5OC, respectively. The pH of the test solutions at test initiation was 7.9 and at test termination ranged from 8.3 to 8.9. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with Selenastrum capricornutum. The light intensity ranged from 3910 to 45 10 lux, which was within the desired range of 4300 +/- 10% lux.

The toxicity of Reofos 65 to Selenastrum capricornutum was determined by evaluating changes in cell density over a 96-hour exposure period. Cell densities were used to calculate areas under the growth curve (biomass) and growth rates for each 24-hour interval of exposure.

After 72 hours of exposure, cell density percent inhibition in the 0.16, 0.3 1, 0.63, 1.3 and 2.5 mg/L treatment groups was 4.5, 21, 30, 25 and 47%, respectively, relative to the negative control.

Percent inhibition for biomass in the 0.16,0.31,0.63, 1.3 and 2.5 mg/L treatment groups was 4.6, 17,27, 2 1 and 43%, respectively, relative to the negative control. Percent inhibition for growth rate in the 0.16, 0.31,0.63, 1.3 and 2.5 mg/L treatment groups was 1.1. 5.1,7.8,6.6 and 14%, respectively, relative to the negative control. Treatment related effects were apparent in the three highest test concentrations
.
Dunnett's test indicated that cell density, biomass and growth rate were significantly reduced (p<0.05) in the 0.63,1.3 and 2.5 mg/L treatment groups. Consequently, the 72-hour NOAEC for cell density, biomass and growth rate was 0.3 1 mg/L.

After 96 hours of exposure, inhibition of cell density in the 0.16, 0.31, 0.63, 1.3 and 2.5 mg/L treatment groups was 3.4, 7.5, 18, 16 and 39%, respectively, relative to the negative control. Percent inhibition of growth rate in the 0.16,0.3 1,0.63, 1.3 and 2.5 mg/L treatment groups was 0.73, 1.5,3.7,3.4 and 9.4%, respectively, relative to the negative control. Treatment related effects were apparent in highest treatment level. While Dunnett's test indicated that cell density and growth rate were significantly reduced ( p < 0.05) the 0.63 and 2.5 mg/L treatment groups the 1.3 mg/L treatment level was not significantly different (p > 0.05). Due to the lack of dose response pattern, the 96-hour NOAEC for cell density and growth rate was 1.3 mg/L.

After 96 hours of exposure, inhibition of biomass in the 0.16, 0.3 1, 0.63, 1.3 and 2.5 mg/L treatment groups was 4.0, 14, 23, 20 and 42%, respectively, relative to the negative control. Treatment related effects were apparent in the three highest test concentrations. Dunnett's test indicated that biomass was significantly reduced ( p < 0.05) in the 0.63,1.3 and 2.5 mg/L treatment groups. Consequently, the 96- hour NOAEC for biomass was 0.3 1 mg/L.

After 96 hours of exposure, there were no signs of adherence of cells to the test chambers or aggregation/flocculation of algae in the controls or in any treatment group. There were no noticeable changes in cell morphology at concentrations 10.63 mg/L when compared to the control. However, some slightly enlarged cells were noted in the 1.3 and 2.5 mg/L treatment groups at 96 hours.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None.
- Unusual cell shape: Slightly enlarged cells were noted in the 1.3 and 2.5 mg/L treatment groups at 96 hours.
- Colour differences: No data.
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: None
Results with reference substance (positive control):
No reference substance was utilised in the study.
Reported statistics and error estimates:
None

Table 1: Measured concentrations of Reefos 65 in Freshwater Algal Medium Samples:

Nominal test concentration

(mg/L)

Sample ID

(298A-121-)

Sampling Time (Hours)

Measured Concentration (mg/L) [1]

Percent of Nominal [2]

Negative Control

1

0

<LOQ

--

0.0

7

96

<LOQ

--

0.16

2

0

0.0801

50.0

0.16

8

96

<LOQ

--

0.31

3

0

0.0887

28.6

0.31

9

96

<LOQ

--

0.63

4

0

0.199

31.6

0.63

10

96

0.0578

9.18

1.3

5

0

0.253

19.4

1.3

11

96

0.0669

5.15

2.5

6

0

0.322

12.9

2.5

12

96

0.216

8.66

[1] The limit of quantitation (LOQ) was 0.0500 mg/L, calculated as the product ofthe concentration of the lowest calibration standard (0.1 00 mg/L) and the dilution factor of the matrix blank samples (0.500).

[2] Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.

Table 2: Temperature Measurements:

Time

(Days)

Temperature (ºC) [1]

Measurement 1

Measurement 2 [2]

0

24.5

23.7

1

23.1

23.0

2

22.7

22.8

3

23.6

23.1

4

23.8

23.7

[1] The minimum and maximum temperature measured continuously throughout the study was 22.8 and 24.5C, respectively.

[2] Temperature Measurement 2 was taken at least 4 hours after Measurement 1, except on Day 4.

Table 3: pH Measurements:

Nominal Test Concentration

(mg/L)

pH Measurements

Day 0 [1]

Day 4 [2]

Negative Control

7.9

8.7

0.16

7.9

8.5

0.31

7.9

8.6

0.63

7.9

8.6

1.3

7.9

8.9

2.5

7.9

8.3

[1] Day 0 samples were collected from the individual batches of test solution prepared for each treatment and control group at test initiation.

[2] Day 4 samples were pooled samples from of test solution collected from each of the replicates per treatment and control group at test termination.

Table 4: Light Intensity Measurements:

Light Intensity (lux) [1]

Test Day

Measurement No. 1

Measurement No.2

Measurement No. 3

Measurement No. 4

Measurement No.5

0

4510

3940

4060

4330

3910

[1] Measurements taken at five locations surrounding the test flasks at test solution level.

Table 5: Mean Cell Density and Percent Inhibition:

Nominal Test Concentration

(mg/L)

24 Hours

48 Hours

72 Hours

96 Hours

Mean Cell Density[1] (Cells/mL)

Percent Inhibition [1,2]

Mean Cell Density[1] (Cells/mL)

Percent Inhibition [1,2]

Mean Cell Density[1] (Cells/mL)

Percent Inhibition [1,2]

Mean Cell Density[1] (Cells/mL)

Percent Inhibition [1,2]

Negative Control

30,137

--

151,847

--

916,498

--

1,927,098

--

0.16

27,424

9.0

147,103

3.1

875,002

4.5

1,861,515

3.4

0.31

26,154

13

146,104

3.8

721,892

21

1,782,127

7.5

0.63

22,343

26

127,777

16

644,811*

30

1,589,676*

18

1.3

26,205

13

145,613

4.1

682,821*

25

1,618,595

16

2.5

21,007

30

110,327

27

487,809*

47

1,175,329

39

[1] Calculations uere performed using SAS Version 8.02. Manual calculations may differ slightly.

[2] Percent inhibition was calculated relative to the negative control replicates.

* Statistically significant difference (p<0.05) from the negative control replicates using Dunnett's test.

Table 6: Mean Area Under the Growth Curve (Biomass) and Percent Inhibition:

Nominal Test Concentration

(mg/L)

0-24 Hours

0-48 Hours

0-72 Hours

0-96 Hours

Mean Area [1]

Percent Inhibition [1,2]

Mean Area [1]

Percent Inhibition [1,2]

Mean Area [1]

Percent Inhibition [1,2]

Mean Area [1]

Percent Inhibition [1,2]

Negative Control

241,648

--

2,185,456

--

14,765,588

--

48,648,740

--

0.16

209,092

13

2,063,420

5.6

14,088,680

4.6

46,686,888

4.0

0.31

193,844

20

2,020,936

7.5

12,196,884

17

42,005,108

14

0.63

148,112

39

1,709,548

22

10,740.600*

27

37,314,436*

23

1.3

194,464

20

2,016,284

7.7

11,717,496*

21

39,094,496*

20

2.5

132,084

45

1,468,092

33

8,405,724*

43

28,123,380*

42

[1] Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.

[2] Percent inhibition was calculated relative to the negative control replicates.

* Statistically significant difference (p4.05) from the negative replicates using Dunnett's test.

Table 7: Mean Growth Rate and Percent Inhibition:

Nominal Test Concentration

(mg/L)

0-24 Hours

0-48 Hours

0-72 Hours

0-96 Hours

Mean Growth Rate [1]

Percent Inhibition [1,2]

Mean Growth Rate [1]

Percent Inhibition [1,2]

Mean Growth Rate [1]

Percent Inhibition [1,2]

Mean Growth Rate [1]

Percent Inhibition [1,2]

Negative Control

0.0454

--

0.0566

--

0.0627

--

0.0548

--

0.16

0.0420

7.5

0.0560

1.1

0.0620

1.1

0.0544

0.73

0.31

0.0396

13

0.0559

1.4

0.0594

5.1

0.0540

1.5

0.63

0.0334

26

0.0531

6.3

0.0577*

7.8

0.0528*

3.7

1.3

0.0401

12

0.0556

1.8

0.0585*

6.6

0.0529

3.4

2.5

0.0306

33

0.0500

12

0.0539*

14

0.0496*

9.4

[1] Calculations were performed using SAS Version 8.02. Manual calculations may differ slightly.

[2] Percent inhibition was calculated relative to the negative control replicates.

* Statistically significant difference (p<0.05) from the negative control replicates using Dunnett's test.

Table 8: EC50, EbC50 and E,C50 Values Over the 96-Hour Exposure Period:

Time

Cell Density

Area Under the Growth Curve (Biomass)

Growth Rate

EC50 (mg/L)

95% Confidence Interval (mg/L)

EbC50 (mg/L)

95% Confidence Interval (mg/L)

ErC50

(mg/L)

95% Confidence Interval (mg/L)

24 Hours

>2.5

--[1]

>2.5

--[1]

>2.5

--[1]

48 Hours

>2.5

--[1]

>2.5

--[1]

>2.5

--[1]

72 Hours

>2.5

--[1]

>2.5

--[1]

>2.5

--[1]

96 Hours

>2.5

--[1]

>2.5

--[1]

>2.5

--[1]

[1] 95% confidence limits could not be calculated with the data obtained.

Validity criteria fulfilled:
yes
Conclusions:
This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at > 5%
The 72 and 96-hour EC50 values for all three parameters were determined to be >2.5 mg/L, the highest concentration of Reefos 65 tested. The 72-hour NOAEC, based on cell density, area under the growth curve and growth rate was determined to be 0.3 1 mg/L. The 96-hour NOAEC, based on the most sensitive endpoint, biomass, was determined to be 0.31 mg/L.
Executive summary:

This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at > 5%.

The 72 and 96-hour EC50 values for all three parameters were determined to be >2.5 mg/L, the highest concentration of Reofos 65 tested. The 72-hour NOAEC, based on cell density, area under the growth curve and growth rate was determined to be 0.31 mg/L. The 96-hour NOAEC, based on the most sensitive endpoint, biomass, was determined to be 0.31 mg/L.

On the basis of the EC50 limit values and the NOECs detailed, it is considered appropriate to classify the substance as "toxic" to aquatic organisms.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2000 – 24 February 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study to recognised study protocols.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Approximately 50 ml of test solution was added to each flask and not 100 ml as stated in the protocol. This protocol deviation does not affect the validity of the study.
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
yes
Details on sampling:
For the purpose of chemical analysis during the definitive test, single aliquots (ca 50 ml) of centrifuged solution were removed from each prepared concentration at the commencement of the test as 0 h samples. At 72 h pooled aliquots (ca 20 ml from each replicate flask including three replicate control flasks) at each concentration were removed as 72 h samples.

An additional flask was prepared at 1000 mg/l and 62.5 mg/l and incubated (without algal cells) alongside test flasks. At 72 h the contents of these flasks were removed for analysis to determine any differences with those flasks containing algae.

Test samples were analysed according to the procedure established and validated under Inveresk Protocol No. 281518.
Vehicle:
no
Details on test solutions:
The following nominal loading rates of Durad 310M in algal medium prepared as water accommodated fraction's (WAF's): 0, 1, 10,100 and 1000 mg/l

Individual WAF's were prepared by adding weighed amounts of Durad 310M (1.04, 9.99,100.16 and 1000.23 mg) to algal medium (1 litre) and stirring this in the dark for an optimal period of 1 h. In the stability trial (conducted under Inveresk Project No. 281518) a period of 1 h stirring was shown to result in the maximum achievable concentration of test item in test medium and the lowest level of degradation. Based on preliminary investigations and observations in the stability trial, WAFs were prepared in 1 litre volumetric flasks and not 2 litre containers as stated in the protocol. This protocol deviation does not affect the validity of the study. The rate of stirring was adjusted to ensure the vortex was <5% of the static depth of the mixture. Following the period of stirring, the flask contents were allowed to settle for 1 h after which ca 350 ml of solution was siphoned from the centre of the vessel, with the first ca 100 ml being discarded. The remaining ca 250 ml of solution was centrifuged (3000 r.p.m. for 15 min) and the supernatant used as the test solution.
Duplicate test flasks were prepared at each concentration, with each flask containing 100 ml of test solution. Two glass marbles were placed in each flask and each flask inoculated with 0.04 x 105 cells.ml/l Pseudokirchneriella subcapitata.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The freshwater green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) was used for this study. Starter cultures (Strain 278/4) were obtained from the Culture Centre of Algae and Protozoa (CCAP), Ambleside, Cumbria, UK on 27 September 2000 (range finding test) and 15 February 2001 (definitive test).

Transfers of the alga were made regularly into fresh algal growth medium to provide suitable axenic subcultures, which were in the exponential phase of growth, for test inoculations
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
Conducted using WAF
Post exposure observation period:
None
Hardness:
Not specified.
Test temperature:
The cell cultures were maintained within an environmental chamber at a temperature of 23-25°C throughout the test period
pH:
7.7 to 7.9 over the range of the study.
Dissolved oxygen:
Not specified.
Salinity:
Not applicable.
Conductivity:
Not specified
Nominal and measured concentrations:
0, 62.5,125, 250, 500 and 1000 mg/l
Details on test conditions:
Test Flasks
Cleaned glass 250 ml Erlenmeyer flasks capped with foil lids were used. Flasks were acid washed and dried at ca 80°C overnight prior to use.

Range Finding Test
A preliminary range finding test was conducted over a 72 h period, using the following nominal loading rates of Durad 310M in algal medium prepared as water accommodated fraction's (WAF's): 0, 1, 10,100 and 1000 mg/l

Individual WAF's were prepared by adding weighed amounts of Durad 310M (1.04, 9.99,100.16 and 1000.23 mg) to algal medium (1 litre) and stirring this in the dark for an optimal period of 1 h. In the stability trial (conducted under Inveresk Project No. 281518) a period of 1 h stirring was shown to result in the maximum achievable concentration of test item in test medium and the lowest level of degradation. Based on preliminary investigations and observations in the stability trial, WAFs were prepared in 1 litre volumetric flasks and not 2 litre containers as stated in the protocol. This protocol deviation does not affect the validity of the study. The rate of stirring was adjusted to ensure the vortex was <5% of the static depth of the mixture. Following the period of stirring, the flask contents were allowed to settle for 1 h after which ca 350 ml of solution was siphoned from the centre of the vessel, with the first ca 100 ml being discarded. The remaining ca 250 ml of solution was centrifuged (3000 r.p.m. for 15 min) and the supernatant used as the test solution.
Duplicate test flasks were prepared at each concentration, with each flask containing 100 ml of test solution. Two glass marbles were placed in each flask and each flask inoculated with 0.04 x 105 cells.ml"1 Pseudokirchneriella subcapitata.

Definitive Test
Based on the results of the range finding test, the definitive test was conducted over a 72 h period at the following nominal loading rates of Durad 310M in medium prepared as WAF's:

0, 62.5,125, 250, 500 and 1000 mg/l

Individual WAF's were prepared by adding weighed amounts of Durad 310M (62.61, 124.97, 250.81, 500.25 and 1000.76 mg) to algal medium (1 litre) and stirring this in the dark for 1 h. The rate of stirring was adjusted to ensure the vortex was <5% of the static depth of the mixture. Following the period of stirring, the flask contents were allowed to settle for 1 h after which ca 600 ml of solution was siphoned from the centre of the vessel, with the first ca 100 ml being discarded. The remaining ca 500 ml of solution was centrifuged (2500 r.p.m. for 15 min) and the supernatant used as the test solution.

Three replicate flasks at each test concentration and six replicate control flasks (algal medium alone) were included in the test. Approximately 50 ml of test solution was added to each flask and not 100 ml as stated in the protocol. This protocol deviation does not affect the validity of the study.

Two glass marbles were placed in each flask and each flask inoculated with 0.09 x 105 cells/ml Pseudokirchneriella subcapitata.

Determination of Durad 310M in Algal Medium
For the purpose of chemical analysis during the definitive test, single aliquots (ca 50 ml) of centrifuged solution were removed from each prepared concentration at the commencement of the test as 0 h samples. At 72 h pooled aliquots (ca 20 ml from each replicate flask including three replicate control flasks) at each concentration were removed as 72 h samples.

An additional flask was prepared at 1000 mg/l and 62.5 mg/l and incubated (without algal cells) alongside test flasks. At 72 h the contents of these flasks were removed for analysis to determine any differences with those flasks containing algae.

Test samples were analysed according to the procedure established and validated under Inveresk Protocol No. 281518.

Monitoring
Algal cell concentrations were measured in the inoculum and in each test flask at 24, 48 and 72 h after the commencement of the definitive test. Aliquots were removed by pipette and cell concentrations were determined using a Compound Light Microscope and Improved Neubauer Counting Chambers. The pH was measured in each prepared concentration at the start and each replicate flask at the end of the definitive test. The pH was measured using a Mettler Toledo MP120 pH meter. Environmental chamber temperature (maximum-minimum) was monitored daily.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: Based on WAF
Details on results:
Range Finding Test
The results indicated that growth was reduced by 92% at 1000 mg/l, 25% at 100 mg/l, 9% at 10 mg/l and 27% at 1 mg/l, as indicated by cell numbers, when compared with control cultures.

Definitive Test
Based on the results of the range finding test, the definitive test was conducted, over a 72 h period, at the following nominal loading rates of Durad 310M in medium: 0, 62.5, 125, 250, 500 and 1000 mg/l

The test solutions were prepared as WAF then centrifuged so that the test algae was exposed only to the water soluble fraction.

Analytical samples were taken at 0 h and 72 h to confirm the concentration of test material in solution. The analytical results indicated that Durad 310M is only partially soluble in algal medium under test conditions with 0 h measured concentrations ranging from 0.240 mg/l to 0.772 mg/l. Analysis of 72 h samples indicated that Durad 310M concentrations were generally maintained over the test period with concentrations ranging from 0.250 mg/l to 0.651 mg/l. The results showed little correlation between measured concentrations of Durad 310M and initial loading rates. This is not uncommon in the preparation of test solutions by the WAF method, with mixtures containing more components than are analysed. No adsorption/desorption to algal cells was indicated by analysis of samples without algae at 72 h.

The average specific growth rate (u ave) was measured for each replicate flask during the experimental period, using daily cell counts. Growth curves were calculated for each test concentration and the area under each curve (AUC) determined.

The following results are based on initial loading rates of Durad 310M:
From AUC data, the EL50 was calculated to be 582 mg/l at 24 h, 415 mg/l at 48 h and 443 mg/lat 72 h. The Pearson chi-square goodness of fit test statistic was statistically significant at the 1% level for all AUC variables due to a non-monotonic increase in percentage inhibition values with concentration. As a result confidence limits are not reported.
Growth rate data did not span 50% inhibition therefore no EL50 values could be calculated. It is concluded that the EL50 for growth rate is >1000 mg Durad 310M / l under the conditions of the test.

The analysis for the NOEL value for AUC (0-24 h) was based on a parametric ANOVA using a log transformation of the data followed by Dunnett's test. The analysis of NOEL value for growth rate (0-72 h) was based on a non-parametric ANOVA based on the ranked data followed by Dunnett's test. The analysis for the remaining NOEL values was based on a parametric ANOVA followed by Dunnett's test, given that Levene's test was not significant at a 1% significance level.

The No Observed Effect Loading (NOEL) could not be determined for AUC or growth rate due to a non-monotonic response. Although the growth parameters in the initial loading rate of 125 mg/l treatment group were not found to be significantly different to the controls, 62.5 mg/l group was significantly different (Table 5). It is noted however that although little correlation was found between effects and mean measured concentration or loading rate, the mean measured concentration at the 125 mg/l loading rate group was the lowest (0.313 mg/l).

The pH of the test solutions remained within protocol specification throughout the test. The temperature of the environmental area was measured daily and remained within the range of 23-25 °C.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
See above

The following results are based on initial loading rates of Durad 310M:

 

Growth Function

Time Interval (h)

EL50Value (mg/l.)

NOEL (mg/l)

Average Specific

0-24

>1000

ND

Growth Rate

0-48

>1000

ND

(Mave.day*1)

0-72

>1000

ND

 

0-24

582

ND

Area Under Growth

0-48

415

ND

Curve (AUC)

 

 

 

 

0-72

443

ND

ND = Not determined

 

Alga Growth Inhibition Test (72 h, EL50) Algal Growth Analysis, EL50Values Based on Average Specific Growth Rates and Areas Under Growth Curves (AUC) During the Definitive Test

 

Growth Function

Time Interval (h)

EL.50Values (mg.r1)

NOEL (mg.r1)

Average Specific Growth Rate(Mave.day1)

0-24

0-48

 0-72

>1000 >1000 >1000

ND ND ND

Area Under Growth Curve (AUC)

0-24

 0-48

0-72

582 415 443

ND ND ND

 

 

Alga, Growth Inhibition Test (72, EL50) Appendix 2    Mean Cell Concentrations (x105cells/ml) at 0, 24, 48 and 72 h During the Definitive Test

 

Time (h)

Replicate/

Nominal Loading of Durad 310M (mq.l*1)

 

Mean

0

62.5

125

250

500

1000

0

Mean

0.09

0.09

0.09

0.09

0.09

0.09

 

1

0.53

0.35

0.75

0.45

0.45

0.38

 

II

0.80

0.35

0.73

0.35

0.33

0.38

 

III

0.83

0.35

0.40

0.30

0.38

0.30

24

IV

0.63

-

-

 

 

 

 

V

0.80

-

-

-

 

_

 

VI

0.53

-

-

-

-

-

 

Mean

0.69

0.35

0.63

0.37

0.39

0.35

 

1

5.08

3.33

5.63

4.65

3.48

0.75

 

II

5.53

3.83

6.43

2.50

3.48

0.50

 

III

6.20

3.65

5.13

3.20

3.38

1.05

48

IV

5.48

-

-

-

-

_

 

V

6.15

-

-

-

-

_

 

VI

5.23

-

-

-

-

-

 

Mean

5.61

3.60

5.73

3.45

3.45

0.77

 

I

21.48

19.33

30.18

25.90

15.95

1.25

 

II

26.93

20.88

26.85

16.08

16.75

1.13

 

III

28.20

19.48

28.73

18.90

17.10

3.55

 

IV

25.63

-

-

-

-

-

 

V

27.08

-

-

-

-

-

 

VI

28.08

-

-

-

-

-

 

Mean

26.23

19.90

28.59

20.29

16.60

1.98

 

Validity criteria fulfilled:
yes
Conclusions:
The 72 h EL50 value of Durad 310M to Pseudokirchneriella subcapitata was determined to be 443 mg/l at 72 h for AUC and >1000 mg/l for growth rate, based on initial loading rates under the conditions of the test.

The No Observed Effect Loading (NOEL) could not be determined due to a non-monotonic response. However, the nominal loading rate of 125 mg/l (0.313 mg/l measured) was not significantly different from the control.
Executive summary:

This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at <5%

The effect of Durad 310M upon the growth of Pseudokirchneriella subcapitata, a freshwater green alga, was determined over a 72 period, in accordance with OECD (1984) Guideline 201.

Test solutions were prepared and tested as water accommodated fractions (WAF) from initial loading rates of 62.5, 125, 250, 500 and 1000 mg/l. A control of untreated algal growth medium was included in the test. The method of preparation was selected to ensure the test material was tested at the maximum achievable solubility under the conditions of the test.

The results are reported as daily cell concentrations (cells/ml), average specific growth rates (day) and areas under growth curves (cells/h/ml). In addition, EL50 values (ie the initial loading rate which results in a 50% reduction in growth) and the no observed effect loading (NOEL) were calculated and are reported in mg/l.

The NOEL for either AUC or average specific growth rate was not determined due to a non-monotonic response. Although the growth parameters in the initial loading rate of 125 mg/l were not found to be significantly different to the controls, 62.5 mg/l group was significantly different. It is noted however that although little correlation was found between effects and mean measured concentration or loading rate, the mean measured concentration at the 125 mg/l loading rate group was the lowest (0.313 mg/l).

The pH of test solutions and the temperature range of the environmental chamber were maintained within protocol specification throughout the definitive test. Illumination was provided by artificial daylight fluorescent tubes giving a light intensity of 9120 lux.

The substance is not considered to be "harmful" to Algae.

Description of key information

Short term toxicity to Algae

Key value for chemical safety assessment

EC50 for freshwater algae:
2.5 mg/L
EC10 or NOEC for freshwater algae:
2.5 mg/L

Additional information

Two studies are presented for growth inhibition to Algae, with the following results:

 

Where TPP content > 5%

EC50 (96 h): > 2.5 mg/L test mat. (nominal) based on: cell density

EC50 (96 h): > 2.5 mg/L test mat. (nominal) based on: area under growth curve

EC50 (96 h): > 2.5 mg/L test mat. (nominal) based on: growth rate

 

Where TPP content < 5%

 

EC50 (72 h): > 1000 mg/L dissolved (nominal) based on: growth rate

 

The substance is not considered to pose a hazard to algae at the limit of solubility in water and is not dependant on triphenyl phosphate content.