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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 July 2016 to 23 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guideline for Testing of Chemicals reference 201 Alga, Freshwater Alga and Cyanobacteria, Growth Inhibition Test reference 201 (2011)
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Lot/Batch Number: NEX-X61-A06 (First batch)*; NEX-X61-A12 (Second batch)**
Expiry Date: 2016-01-09 (First batch)*; 29th April 2017 (Second batch)**
*The first batch was used in the range-finding test.
**The second batch was used in the limit test and full definitive test.
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken at the start and end of the 72 hour exposure period. Samples were taken from remaining test media after filling test vessels for 0 hours. At 72 hours replicate vessels were pooled and centrifuged, to remove the algal cells, with samples taken from the supernatant.
Vehicle:
no
Details on test solutions:
Information provided by the Sponsor stated the sample was insoluble in water.
For the range-finding test a water accommodated fraction (WAF) of 100mg/l was prepared by adding 0.0502g of the test substance to algal medium in an aspirator. This was stirred for 46 hours and then left to settle for 4 hours and 15 minutes. After settling the aqueous phase was drawn off for testing leaving any sedimented/undissolved material behind. The solution was filtered , with the initial 150ml of filtrate discarded. The lower doses (0.1, 1.0 and 10mg/l) were then prepared by dilution of the 100mg/l aqueous phase with algal medium, this was considered to be the most practical method for the low concentrations required.
Since no inhibition was seen in the range-finding test at 100mg/l, which actually had a slightly stimulated growth response than that of the unexposed control, a limit test was carried out. The test concentration (100mg/l) for the definitive limit test was prepared analogously to the stock solution in the range-finding test. A WAF was prepared by adding 0.1007g of the test substance to algal medium in an aspirator. This was stirred for 69 hours and 25 minutes and then left to settle for 25 hours and 55 minutes. After settling the aqueous phase was drawn off for testing avoiding any sedimented/undissolved material. The solution was filtered using a Whatman 54 filter, with the initial 100ml of filtrate discarded.
As the reduction in growth rate in the limit test was statistically signifcant (at P=0.05) compared to the unexposed control, a full definitive test was carried out. A WAF of 200mg/l was prepared by adding 0.2000g of the test substance to algal medium in an aspirator. This was stirred for 70 hours and 15 minutes and then left to settle for 4 hours and 30 minutes. After settling the aqueous phase was drawn off for testing avoiding any sedimented/undissolved material. The solution was filtered using a Whatman 54 filter, with the initial 100ml of filtrate discarded. The lower test concentrations (12.5, 25, 50 and 100mg/l) were then prepared by dilution of the 200mg/l aqueous phase, with algal medium, volumetrically.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism
Species: Pseudokirchneriella subcapitata strain: CCAP 278/4.
received 22 April 2016.
Source: Culture Collection of Algae and Protozoa, SAMS Research Services Ltd; Scottish Marine institute, OBAN, Argyll PA37 1QA, Scotland, United Kingdom
Culture conditions: Temperature: 21.2-21.4C.
Illumination: 6020-7720 lux continuous white light.
Orbiting: set to 200rpm.
Culture media: Deionised water with added nutrients according to OECD 201.

Test and culture medium: The culture medium and the control medium were prepared with deionised water (sterilised by autoclaving at 120°C for 15 minutes) with added nutrients. The pH of the control medium was adjusted to 8.05 ± 0.1.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not specified
Test temperature:
Temperature range within incubator throughout test: 21.2-22.1°C
(Required: 21-24±2°C)
pH:
Initial pH at 0 Hours: 8.05 (Adjusted from 7.68)
(Required: 8.1±0.1)
Dissolved oxygen:
Not specified
Salinity:
Not applicable - freshwater study
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal: 0 (control), 12.5, 25, 50, 100 and 200mg/l
Details on test conditions:
The test was carried out according to the procedures given below, based on the guidelines produced by OECD 201: Alga Growth Inhibition Test.
Chemex SOP reference: E203 Algal Growth Inhibition Test (Freshwater)
Preliminary test method: A preliminary (range finding) test was conducted at concentrations of 0 (control), 0.1, 1.0, 10, 100mg/l. The duration of the preliminary study was 72 ± 2 hours. There was a single replicate at each concentration.
Preliminary test results: No inhibition was seen in the preliminary test. The growth rate at the highest test concentration (100mg/l) exhibited a stimulated growth response compared to that of the unexposed control. Therefore, the 72 hour EL50 (by growth rate) was >100mg/l.

Definitive test method:
Test period: 20 to 23 February 2017
Test duration: 72 ± 2 hours
Test volume: 100ml
Test vessel: 250ml conical flask
Number of replicates: Six control flasks, three replicates at each test concentration.
Test concentrations: 0 (control), 12.5, 25, 50, 100 and 200mg/l
Algal Test innoculum: From a pre-culture growing in exponential phase. Inoculum level adjusted to give an initial cell density of 1 x 104 cells/ml.

Composition of medium: Sterilised deionised water with added nutrients according to the OECD 201 standard.

Test conditions: Initial pH at 0 Hours: 8.05 (Adjusted from 7.68) (Required: 8.1±0.1)
Temperature range within incubator throughout test: 21.2-22.1°C (Required: 21-24±2°C)
Illumination range within incubator throughout test: 6120 –7720 Lux (Required: 6x103-8x103 Lux ± 15% of recorded mean)
Orbital shaking: 200rpm (Required: 200-250rpm)

Water quality measurements: The temperature (to 0.1°C) and light intensity (lux) within the incubator was recorded at the beginning of the study, after 24, 48 hours and at the end of the 72-hour test period. The pH (to 0.01) and temperature (to 0.1°C) were recorded for each test and control solution at the beginning of the test and on the pooled replicates at the end of the 72-hour test period.

Observations/frequency: Cell densities were measured microscopically by direct cell counts on each test and control replicate, in sextuplicate at 24 hours for the control and triplicate thereafter, and in triplicate at 24, 48 and 72 hours (±2h) for the test concentrations. The cell counts were made using a haemocytometer and microscope.

Calculation of results: Growth curves for each test concentration were plotted as logarithm of the mean cell density against time. The inhibition of growth was calculated using both the biomass (increase in cell yield) and the growth rate method. Where possible, the EL50 values were estimated graphically and 95% confidence limits calculated according to the method of ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”, copyright 1994-1996.

Reference Substance: A separate GLP reference test was performed on potassium dichromate outside of this study as a means of checking the test procedure and sensitivity of the test species (ENV 11396 from 16 to 19 January 2017).
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
140.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: All concentrations are expressed as loading rates from water-accommodated fractions (WAFs)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
158.58 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: All concentrations are expressed as loading rates from water-accommodated fractions (WAFs)
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
Remarks on result:
other: All concentrations are expressed as loading rates from water-accommodated fractions (WAFs)
Details on results:
The algal cells were examined microscopically during the determination of the cell density, all cells within the control and all test concentrations appeared normal, no morphological abnormalities were observed.
When observed visually, the control conical flasks and those for test concentrations 12.5, 25, 50 and 100mg/l appeared clear and colourless up until 48 hours when they were recorded as being pale green. They were then observed as being green in colour after 72 hours. The conical flasks for the highest test concentration (200mg/l) appeared very pale yellow at 0 and 24 hours. After 48 hours they were recorded as being pale green/yellow and pale yellow at 72 hours.
Results with reference substance (positive control):
ISO 8692:2012 quotes the 72 hour EC50 for potassium dichromate for growth rate as 1.19mg/l (mean value; standard deviation 0.27; based upon ring tests) and therefore the result obtained in the reference test should be in the range 0.65 and 1.73mg/l (mean value ± 2 x standard deviation).
Reported statistics and error estimates:
The 72 hour EL(r)50 & EL(y)50 of Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product to Pseudokirchneriella subcapitata were 155.8mg/l (determined by Maximum Likelihood-Logit) and 140.4mg/l (determined by Maximum Likelihood-Weibull), respectively.
The 0 to 72-hour NOELR(r) was 100mg/l and the 0-72 hour LOELR(r) was 200mg/l (both determined by Bonferroni t Test).

Preliminary test results:

Nominal conc.

(mg/l)

Percent inhibition by yield

Percent inhibition by growth rate

0 – 72 hours

0 – 72 hours

0.1

15

4

1.0

2

0

10

-9

-2

100

-74

-14

Note: Negative numbers indicate an increase in growth compared to control data.

All concentrations are expressed as loading rates from water-accommodated fractions.

 

Cell density measurements

Mean initial cell density: Approximately 1 x 104 cells/ml based upon inoculation volume, not counted microscopically.

Nominal concentration

(mg/l)

Replicate

Cell density measurements (cells/ml x 104)

24 hours

48 hours

72 hours

0 (Control)

1

2.0

13.3

59.7

2

3.5

24.63

127.7

3

4.0

34.0

115.7

4

6.0

26.3

101.0

5

4.0

21.7

127.3

6

4.2

22.0

116.0

Mean

4.0

23.6

107.9

12.5

1

3.3

21.3

96.3

2

2.7

20.7

128.3

3

3.3

31.0

97.7

Mean

3.1

24.3

107.4

25

1

5.7

22.0

98.7

2

3.3

29.0

122.7

3

6.7

27.3

110.0

Mean

5.2

26.1

110.5

50

1

3.7

17.3

94.3

2

6.3

19.7

105.3

3

2.7

19.7

127.7

Mean

4.2

18.9

109.1

100

1

5.3

26.0

90.7

2

3.3

11.3

108.7

3

4.7

13.3

94.0

Mean

4.4

16.9

97.8

200

1

1.3

3.0

1.7

2

1.7

2.3

2.0

3

1.3

1.0

1.0

Mean

1.4

2.1

1.6

 

Percent inhibition

Nominal concentration

(mg/l)

Percent inhibition by yield

Percent inhibition by growth rate

0 – 48 hours

0 – 72 hours

0 – 48 hours

0 – 72 hours

12.5

-3

0

-2

0

25

-11

-2

-4

-1

50

21

-1

6

-1

100

30

9

12

2

200

95

99

79

91

 

Water qualities and incubation conditions

Concentration

(mg/l)

0 hours

72 hours

pH

Temp (°C)

pH

Temp (°C)

0 (Control)

8.05

21.2

7.71

21.4

12.5

8.00

21.2

7.00

21.2

25

8.06

21.2

6.89

21.4

50

8.09

21.1

6.80

21.3

100

8.11

21.0

6.70

21.5

200

8.23

21.0

5.72

21.4

 

Incubator temperature and light intensity measurements

Within incubator

Temperature (°C)

0 hours

24 hours

48 hours

72 hours

Current

21.2

21.3

21.6

21.5

Minimum

 

21.3

21.3

21.2

Maximum

 

21.3

21.7

21.6

 

Within incubator

Light intensity (lux)

0 hours

24 hours

48 hours

72 hours

Min

6120

6210

6550

6360

Max

7720

7030

7180

7660

 

Validity criteria fulfilled:
yes
Conclusions:
The 72 hour EL(r)50 & EL(y)50 of Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product to Pseudokirchneriella subcapitata were 155.8mg/l and 140.4mg/l, respectively.
The 0 to 72-hour NOELR(r) was 100mg/l and the 0-72 hour LOELR(r) was 200mg/l.
The algal cells were examined microscopically during the determination of cell concentration. All cells within the control and test concentrations appeared normal, no abnormalities were observed.
Executive summary:

Substance under test: Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product

Chemex reference: Sample: ECO150401

Study: ENV10803

Test species: Pseudokirchneriella subcapitata, strain CCAP 278/4

Test type: Growth inhibition: 72-hour EL50

Protocol: Test conditions according to SOP E203 based upon OECD Guideline for testing of chemicals, Freshwater Alga and Cyanobacteria, Growth Inhibition Test reference 201 (2011).

Experimental period: Range-finding test: 15 to 22 July 2016

Definitive limit test: 26 January to 03 February 2017

Full definitive test: 17 to 23 February 2017

Test concentrations: 0 (control), 12.5, 25, 50, 100 and 200mg/l

Test performed at: Chemex Environmental International Limited

Results: All study validity criteria were met. The results are summarised in the table below.

 

Exposure Period (hours)

EyLxvalue mg/l

(95% confidence limits)

ErLxvalue (mg/l)

(95% confidence limits)

EyL10

EyL20

EyL50

ErL10

ErL20

ErL50

0 to 48

67.48(1)

(44.98-80.58)

82.74(1)

(63.47-94.50)

117.2(1)

(104.2-136.2)

95.26(1)

(73.50-110.8)

112.9(1)

(92.90-127.5)

151.0(1)

(135.1-166.4)

0 to 72

101.1(2)

(91.27-109.7)

115.2(2)

(106.3-129.3)

140.4(2)

(126.0-176.0)

123.3(1)

(91.35-141.2)

134.4(1)

(105.1-150.5)

155.8(1)

(133.2-168.2)

NOELR

(0-72h)

100 mg/l

(Determined by Bonferroni t Test)*

100 mg/l

(Determined by Bonferroni t Test)*

(1)Determined by Maximum Likelihood-Logit, using ToxCalc v5.0

(2)Determined by Maximum Likelihood-Weibull, using ToxCalc v5.0

*Following Shapiro-Wilk’s test for normality of distribution and Bartlett’s test for equal variances.

All concentrations are expressed as loading rates from water-accommodated fractions (WAFs)

Description of key information

The substance is known to hydrolyse relatively rapidly in water, hence both the substance and the associated hydrolysis product were assessed within the test.

The 72 hour EL(r)50 & EL(y)50 of Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product to Pseudokirchneriella subcapitata were 155.8mg/l and 140.4mg/l, respectively.

The 0 to 72-hour NOELR(r) was 100mg/l and the 0-72 hour LOELR(r) was 200mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:
140.4 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Substance under test: Phenylacetylene modified trimellitic anhydride (PETA) including the hydrolysis product

Test species: Pseudokirchneriella subcapitata, strain CCAP 278/4

Test type: Growth inhibition: 72-hour EL50

Protocol: Test conditions according to SOP E203 based upon OECD Guideline for testing of chemicals, Freshwater Alga and Cyanobacteria, Growth Inhibition Test reference 201 (2011).

Test concentrations: 0 (control), 12.5, 25, 50, 100 and 200mg/l

Results: All study validity criteria were met. The results are summarised in the table below.

 

Exposure Period (hours)

EyLxvalue mg/l

(95% confidence limits)

ErLxvalue (mg/l)

(95% confidence limits)

EyL10

EyL20

EyL50

ErL10

ErL20

ErL50

0 to 48

67.48(1)

(44.98-80.58)

82.74(1)

(63.47-94.50)

117.2(1)

(104.2-136.2)

95.26(1)

(73.50-110.8)

112.9(1)

(92.90-127.5)

151.0(1)

(135.1-166.4)

0 to 72

101.1(2)

(91.27-109.7)

115.2(2)

(106.3-129.3)

140.4(2)

(126.0-176.0)

123.3(1)

(91.35-141.2)

134.4(1)

(105.1-150.5)

155.8(1)

(133.2-168.2)

NOELR

(0-72h)

100 mg/l

(Determined by Bonferroni t Test)*

100 mg/l

(Determined by Bonferroni t Test)*

(1)Determined by Maximum Likelihood-Logit, using ToxCalc v5.0

(2)Determined by Maximum Likelihood-Weibull, using ToxCalc v5.0

*Following Shapiro-Wilk’s test for normality of distribution and Bartlett’s test for equal variances.

All concentrations are expressed as loading rates from water-accommodated fractions (WAFs)