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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 16 July, 2012 to 09 Aug, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
according to
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
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, t=24 h and t=72 h
Volume: 1.5 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, after 24 h of exposure and at the end of the test period. Additionally, reserve samples of 1.5 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:
Ebecryl 140 radiation curing resins, a UVCB substance, was not completely soluble in test medium at the loading rates initially prepared. Weighing of test substance was performed under either dimmed or yellow light. Preparation of test solutions was performed under dimmed light conditions. 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. WAFs were observed to range from clear and colourless to hazy with increasing loading rate. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 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.
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.
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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
Between 22.5 and 22.8°C
pH:
Between 8.0 - 8.4
Nominal and measured concentrations:
Nominal concentrations: WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L
Measured concentrations: Analysis of the samples taken at the start of the final test showed measured concentrations of 0.42, 2.2, 2.6, 19 and 27 mg/L at the WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. The three lowest test concentrations remained stable during the first 24 h of exposure (82-103% of initial) but decreased to 66-87% of initial at the end of the test. The two highest test concentrations decreased to 35-61% of initial during the first 24 h of exposure but remained stable at 38-59% of initial during the remainder of the test. Based on these results, the average exposure concentrations were calculated to be 0.35, 1.7, 2.5, 12 and 12 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 64 to 73 µE.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 extra replicate of the control and each test concentration for sampling purposes; 2 replicates of the highest test concentration without algae.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
72 h NOErC, 72 h NOEyC, 72 h ErC50, 72 h EyC50, 72h-NOErC, 72h-NOEyC

Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.35 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.51 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.35 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.35 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Microscopic observations during the test revealed a normal and healthy appearance of the exposed cells when compared to the control
Results with reference substance (positive control):
EC50 for growth rate reduction was 2.3 mg/L with a 95% confidence interval ranging from 1.9 to 2.9 mg/L
EC50 for yield inhibition 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: 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 average exposure concentrations of the test substance.

Combined limit/range-finding test:

Based on the mean cell densities measured and the percentages growth rate reduction and yield inhibition per concentration, samples taken from the WAFs prepared at loading rates of 1.0 and 100 mg/L were analysed. The initial concentrations were 0.61 and 37 mg/L, respectively. These concentrations decreased to 5-74% of initial after 24 h of exposure and further to below the lowest calibration solution for the 1.0 mg/L WAF and to 50% of initial for the 100 mg/L WAF at the end of the test. The expected EC50 for growth rate reduction was above a Time Weighted Average (TWA) concentration of 26 mg/L. The expected EC50 for yield inhibition was between TWA concentrations of 0.056 and 26 mg/L. All test conditions were maintained within the limits prescribed by the protocol.

 

Final test

-Measured test substance concentrations: Analysis of the samples taken at the start of the final test showed measured concentrations of 0.42,2.2, 2.6, 19 and 27 mg/L at the WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. Thethree lowest test concentrations remained stable during the first 24 h of exposure (82-103% ofinitial) but decreased to 66-87% of initial at the end of the test. The two highest test concentrationsdecreased to 35-61% of initial during the first 24 h of exposure but remained stable at 38-59% ofinitial during the remainder of the test. Based on these results, the average exposure concentrationswere calculated to be 0.35, 1.7, 2.5, 12 and 12 mg/L.

-Mean cell densities:

Table 1. Mean cell densities (x 104cells/mL) during the final test

Loading rats ebecryl® 140

radiation curing

resins (mg/L)

Exposure time (h)

0

24

48

72

Control

1.0

3.0

16.0

78.8

1.0 (0.35)

1.0

2.4

12.0

52.2

3.2 (1.7)

1.0

2.3

9.6

37.3

10 (2.5)

1.0

2.5

8.4

34.3

32 (12)

1.0

1.3

6.2

20.8

100 (12)

1.0

2.0

3.8

13.7

 

-Reduction of growth rate and inhibition of yield: Statistically significant reduction of growth rate and inhibition of yield was found at all test concentrations (Bonferroni t-Test, α= 0.05). Microscopic observations during the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

 

Table 2. Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Loading rats ebecryl® 140

radiation curing

resins (mg/L)

Mean growth rate

Yield (0-72 h)

μ (0-72 h)

Reduction (%)

x104cells/mL

Inhibition (%)

Control

0.06062

 

77.83

 

1.0 (0.35)

0.05480

9.6

51.17

34.3

3.2 (1.7)

0.05010

17.3

36.25

53.4

10 (2.5)

0.04906

19.1

33.25

57.3

32 (12)

0.04193

30.8

19.75

74.6

100 (12)

0.03605

40.5

12.67

83.7

 

-Determination of effect concentrations:

Table 3. Effect parameters

Parameter

Concentration

ebecryl® 140

radiation curing

resins (mg/L)

95%-confidence

interval

72h-NOERC

<0.35

 

72h-ERC10

0.51

0.10 - 2.7

72h-ERC50

>12

 

72h-NOEYC

<0.35

 

72h-EYC10

<0.35

 

72h-EYC50

1.3

0.38- 4.3

 

- Experimental conditions: The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). The temperature of the test medium was 21.6°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.5 and 22.8°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the 72 h EC50 and EC10 values of ebecryl 140 in algae for growth rate reduction were > 12 and 0.51 mg/L, respectively. The EC50 for yield inhibition was 1.3 mg/L with a 95% confidence interval ranging from 0.38 to 4.3 mg/L. Further, the NOEC for both growth rate reduction and yield inhibition was < 0.35 mg/L.
Executive summary:

A study was performed to assess the acute toxicity of ebecryl 140 to the algae, Pseudokirchneriella subcapitata according to the OECD Guideline 201. In addition, the procedures were designed to meet the test methods of EU Part C.3, 2008, the ISO International Standard 8692, 2004 and the OECD series on testing and assessment number 23, 2000, in compliance with GLP. Three replicates of exponentially growing algal cultures were exposed to water accommodated fractions (WAFs) prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. In addition, six replicates were exposed to a control. The initial cell density was 104 cells/mL and the total test period was 72 h. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 h of exposure and at the end of the test. Analysis of the samples taken at the start of the final test showed measured concentrations of 0.42, 2.2, 2.6, 19 and 27 mg/L for the WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, respectively. The three lowest test concentrations remained stable during the first 24 h of exposure (82-103% of initial) but decreased to 66-87% of initial at the end of the test. The two highest test concentrations decreased to 35-61% of initial during the first 24 h of exposure but remained stable at 38-59% of initial during the remainder of the test. Based on these results, the average exposure concentrations were calculated to be 0.35, 1.7, 2.5, 12 and 12 mg/L. Under the test conditions, the test substance reduced growth rate and inhibited the yield of this fresh water algae species significantly at all concentrations tested. The EC50 and EC10 for growth rate reduction were > 12 and 0.51 mg/L, respectively. The EC50 for yield inhibition was 1.3 mg/L with a 95% confidence interval ranging from 0.38 to 4.3 mg/L. The NOEC for both growth rate reduction and yield inhibition was < 0.35 mg/L (Bouwman, 2013).

Description of key information

Based on the study results, the 72 h ErC50 and ErC10 values of the test substance in algae were >12 and 0.51 mg/L (measured), respectively. The 72 h EbC50 was 1.3 mg/L with a 95% confidence interval ranging from 0.38 to 4.3 mg/L. Further, the 72 h NOEC for both growth rate reduction and yield inhibition was <0.35 mg/L (measured).

Key value for chemical safety assessment

EC50 for freshwater algae:
12 mg/L
EC10 or NOEC for freshwater algae:
0.51 mg/L

Additional information

A study was performed to assess the acute toxicity of the test substance to the algae Pseudokirchneriella subcapitataa ccording to OECD Guideline 201, Commission Regulation (EC) No 440/2008, Part C.3, 2008 (amended by EC No. 761/2009) and ISO International Standard 8692, 2004. Three replicates of exponentially growing algal cultures were exposed to WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. In addition, six replicates were exposed to a control. The initial cell density was 104cells/mL and the total test period was 72 h. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 h of exposure and at the end of the test. Analysis of the samples taken at the start of the final test showed measured concentrations of 0.42, 2.2, 2.6, 19 and 27 mg/L for the WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, respectively. The three lowest test concentrations remained stable during the first 24 h of exposure (82-103% of initial) but decreased to 66-87% of initial at the end of the test. The two highest test concentrations decreased to 35-61% of initial during the first 24 h of exposure but remained stable at 38-59% of initial during the remainder of the test. Based on these results, the average exposure concentrations were calculated to be 0.35, 1.7, 2.5, 12 and 12 mg/L. Under the test conditions, the test substance reduced growth rate and inhibited the yield of this freshwater algae species significantly at all tested concentrations. The 72 h ErC50 and ErC10 for growth rate reduction were >12 and 0.51 mg/L, respectively. The 72 h EbC50 for yield inhibition was 1.3 mg/L with a 95% confidence interval ranging from 0.38 to 4.3 mg/L. The 72 h NOEC for both growth rate reduction and yield inhibition was <0.35 mg/L.