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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:
From 25 June 2012 to 1 Aug, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: At t=0 h and t=72 h
Volume : 2 mL
Storage : Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae, and samples for analysis were taken at the start and at the end of the test period. Additionally, reserve samples of 2 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:
no
Details on test solutions:
DPHA, a UVCB substance, was not completely soluble in test medium at the loading rates initially prepared. Weighing of test substance and preparation of test solutions was performed under either dimmed light. All test concentrations were prepared separately applying 2 d of magnetic stirring in the dark to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were left to stabilize for 3 h where after the Water Accommodated Fractions (WAFs) were siphoned off and used as test concentrations. All WAFs were observed to be clear and colourless, except for the 100 mg/L WAF which was observed to be slightly hazy. After preparation, volumes of 50 mL were added to each replicate of the respective test concentrations. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10000 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
Pre-culture: 3 d before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10000 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/sec when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
At the end of the final test microscopic observations were performed on the two highest test concentrations to observe for any abnormal appearance of the algae.
Hardness:
24 mg CaCO3/L
Test temperature:
Between 22.7 and 23.3
pH:
Between 8.1 and 8.4
Nominal and measured concentrations:
Nominal concentrations:WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L
Measured concentrations:The quantitative analysis of the test substance in water was based on the response of the two major components DPPA and DPHA. Samples taken from the WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L were analysed. The initial concentrations were 9.0, 16, 30, 50 and 88 mg/L, respectively when based on DPPA and 6.6, 12, 20, 26 and 36 mg/L, respectively when based on DPHA. These concentrations remained stable during the test period (97-102% of initial for DPPA and 84-89% of initial for DPHA at the end of the test). Given these results, the effect parameters could be based on initial exposure concentrations. Following a worst case scenario DPHA concentrations were used to calculate the effect parameters, i.e. 6.6, 12, 20, 26 and 36 mg/L.
Details on test conditions:
Test duration: 72 h
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1 x 10E4 cells/mL.
Illumination: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 62 to 71 uE.m-2.sec-1.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking
Controls: Test medium without test substance or other additives
Replicates:
3 replicates of each test concentration;
6 replicates of the control;
1 or 2 replicates of each test concentration without algae.
Effect concentrations:
72h-NOERC
72h-ERC10
72h-ERC50
72h-NOEYC
72h-EYC10
72h-EYC50

Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7,)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 6.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval 6.3-26 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 36 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 6.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 6.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence interval 3.3 - 14 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 21 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence interval 11-41 mg/L
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
Behavioural abnormalities: No
- Immobility of control: No immobility was observed.
- Other adverse effects control: No
Results with reference substance (positive control):
The EC50 for growth rate reduction (ERC50: 0-72h) was 2.3 mg/L with a 95% confidence interval ranging from 1.9 to 2.9 mg/L.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.90 mg/L with a 95% confidence interval ranging from 0.68 to 1.2 mg/L.
Reported statistics and error estimates:
Determination of the NOEC and calculation of the EC50: For determination of the NOEC and the EC50 the approaches recommended in OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-Test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding initial exposure concentrations of the test substance.

Table 1. Mean cell densities (x104cells/mL) during the final test:

Loading rate1DPHA

(mg/L)

Exposure time (h)

 

 

0

24

48

72

Control

1.0

6.2

24.3

89.5

10 (6.6)

1.0

6.4

22.8

78.8

18(12)

1.0

6.2

19.3

61.9

32 (20)

1.0

5.9

18.6

57.8

56 (26)

1.0

5.3

13.3

34.3

100 (36)

1.0

5.7

11.5

24.8

1WAF prepared at the given loading rate

Table 2. Percentage reduction of growth rate and inhibition of yield during the final test

Loading rate1DPHA

(mg/L)

Mean growth rate

 

 

Yield (0-72 h)

 

 

 

μ (0-72 h)

Reduction (%)

x104cells/mL

Inhibition (%)

Control

0.06236

 

88.51

 

10 (6.6)

0.06045

3.1

77.78

12.1

18(12)

0.05729

8.1

60.95

31.1

32 (20)

0.05630

9.7

56.82

35.8

56 (26)

0.04907

21.3

33.28

62.4

100 (36)

0.04458

28.5

23.82

73.1

1WAF prepared at the given loading rate

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, 72 h ErC50, EyC50 and NOEC for the test substance were determined to be >36, 21 and 6.6 mg/L (measured initial, WAF), respectively.
Executive summary:

A study was conducted to determine the acute toxicity of the test substance, DPHA, to the green algae, Pseudokirchneriella subcapitata, according to OECD Guideline 201, EU Method C.3 and ISO Standard 8692, in compliance with GLP. Three replicates of exponentially growing algal cultures were exposed to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. In addition, six replicates were exposed to a control. The initial cell density was 1E+4 cells/mL and the total test period was 72 h. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. The quantitative analysis of the test substance in water was based on the response of the two major components DPPA and DPHA. Samples taken from the WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L were analysed. The initial concentrations were 9.0, 16, 30, 50 and 88 mg/L, respectively, when based on DPPA and 6.6, 12, 20, 26 and 36 mg/L, respectively, when based on DPHA. These concentrations remained stable during the test period (97-102% of initial for DPPA and 84-89% of initial for DPHA at the end of the test) and did not deviate more than 20% from the nominal loading rates. Given these results, the effect parameters could be based on initial exposure concentrations or based on initial loading rates. The study met the acceptability criteria prescribed by the protocol and was considered valid. DPHA reduced growth rate and inhibited the yield of this fresh water algae species significantly at 12 mg/L and higher. The EC50 for growth rate reduction (ERC50: 0-72h) was >36 mg/L (corresponding to EL50 of 100 mg/L). The EC50 for yield inhibition (EYC50: 0-72h) was 21 mg/L with a 95% confidence interval ranging from 11 to 41 mg/L. The NOEC for both growth rate reduction and yield inhibition was 6.6 mg/L (equivalent to a loading rate of 10 mg/L). Under the study conditions, 72 h ErC50, EyC50 and NOEC for the test substance were determined to be >36 (corresponding to EL50 of 100 mg/L), 21 and 6.6 mg/L (equivalent to NOE(L)C of 10 mg/L), respectively (Bouwman, 2013).

Description of key information

In the short term toxicity study to aquatic algae, the initial measured concentrations were 9.0 mg/L (DPPA) and 7.2 mg/L (DPHA) at the 10 mg/L WAF and 16 mg/L (DPPA) and 11 mg/L (DPHA) at the 18 mg/L WAF. These concentrations remained stable during the test period (102-105% of initial for DPPA and 87-94% of initial for DPHA at the end of the test). Overall, the deviation between the nominal loading rates and the measured concentrations is less than 20%. Given these results, the effect parameters could be based on initial measured concentrations or even based on the initial loading rates (WAF). In absence of information on the actual moiety driving the toxicity, it was decided to base the effect concentrations on the initial loading rates. Therefore, the EL50 and NOE(L)C for growth inhibition of aquatic algae were considered to be 100 mg/L and 10 mg/L respectively (equivalent to the measured concentrations of 36 mg/L and 6.6 mg/L respectively).

Key value for chemical safety assessment

EC50 for freshwater algae:
36 mg/L
EC10 or NOEC for freshwater algae:
6.6 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance, DPHA, to the green algae, Pseudokirchneriella subcapitata, according to OECD Guideline 201, EU Method C.3 and ISO Standard 8692, in compliance with GLP. Three replicates of exponentially growing algal cultures were exposed to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. In addition, six replicates were exposed to a control. The initial cell density was 1E+4 cells/mL and the total test period was 72 h. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. The quantitative analysis of the test substance in water was based on the response of the two major components DPPA and DPHA. Samples taken from the WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L were analysed. The initial concentrations were 9.0, 16, 30, 50 and 88 mg/L, respectively, when based on DPPA and 6.6, 12, 20, 26 and 36 mg/L, respectively, when based on DPHA. These concentrations remained stable during the test period (97-102% of initial for DPPA and 84-89% of initial for DPHA at the end of the test) .Overall, the deviation between the nominal loading rates and the measured concentrations is less than 20%. Given these results, the effect parameters could be based on initial exposure concentrations or based on initial loading rates.  The study met the acceptability criteria prescribed by the protocol and was considered valid. DPHA reduced growth rate and inhibited the yield of this fresh water algae species significantly at 12 mg/L and higher. The EC50 for growth rate reduction (ERC50: 0-72h) was >36 mg/L (corresponding to EL50 of 100 mg/L). The EC50 for yield inhibition (EYC50: 0-72h) was 21 mg/L with a 95% confidence interval ranging from 11 to 41 mg/L. The NOEC for both growth rate reduction and yield inhibition was 6.6 mg/L (equivalent to a loading rate of 10 mg/L). Under the study conditions, 72 h ErC50, EyC50 and NOEC for the test substance were determined to be >36 (corresponding to EL50 of 100 mg/L), 21 and 6.6 mg/L (equivalent to NOE(L)C of 10 mg/L), respectively (Bouwman, 2013).