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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:
2013-12-16 until 2014-01-16
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all (At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.)
- Sampling frequency: at t=0 h, t=24 h and t=72 h
- Sample volume: 2.0 mL
- Sample storage conditions before analysis: stored in a freezer until analysis (<= -15°C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with a nominal concentration of 100 mg/l. Fifteen minutes of magnetic stirring was applied to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 100 mg/l concentration in test medium. Test solutions up to and including 32 mg/l were clear and colourless, while the higher test concentration of 100 mg/l was clear and slightly yellow/brown.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchnerella subcapitata
- Source (laboratory, culture collection): in-house laboratory culture
- Age of inoculum (at test initiation): 2-4 d
- Method of cultivation: Algae stock cultures are started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions are continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. Light intensity is 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

PRE-CULTURE
- Culturing media and conditions (same as test or not):yes
- Cell density: 1 x 10^4 cells/mL
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L (M2 medium)
Test temperature:
21.2 - 23.0 °C
pH:
7.2 - 8.3
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 mL of test solution
- Type: capped vessels, continous shaking
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 1x 10^4 cells/mL
- Control end cells density: average increase factor of >16 within two days
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
-Other: 1 extra replicateof the control and each test concentration for sampling purposes; 2 replicates of the 3.2 mg/L concentration without algae

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water
- Pre-culture medium different from test medium: no
- Intervals of water quality measurement: at 0 hours and at 72 hours

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous (TLD-lamps)
- Light intensity and quality: 87 to 96 µE.m^-2s^-1
- Salinity (for marine algae): not applicable
-Incubation: Incubator with daily repositioning

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscope and counting chamber
- Other: At the end of the final test microscopic observations were performed on all test concentrations and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: not applicable
- Range-finding test concentrations [mg/L]: 0.1, 1.0, 10, 100
- Test concentrations [mg/L]: 0.10, 0.32, 1.0, 3.2, 10, 32, 100
- Results used to determine the conditions for the definitive study (inhibition in %): 0.9% at 0.1 mg/L, 23.2 % at 1.0 mg/L, 51.2 % inhibition at 10 mg/L
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 2.2 - 9.1 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
42 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 27 - 72 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 99 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
Parameters of probit analysis (growth rate)
---------------------------------------
Computation runs: 9.0000
Slope b: 0.4835
Intercept a: -1.62849
Variance of b: 0.5048
Goodness of Fit
Chi²: 0.1791
Degrees of freedom: 19.0000
p(Chi²): 1.0000
Log EC50: 3.3682
SE Log EC50: 2.9992
g-Criterion: 0.0892
Residual Variance (Chi²/df): 0.0094
r²: 0.7210
F: 49.1330
p(F) (df: 1;19): 0.0000

Results of the probit analysis (growth rate)

Parameter:              EC10       EC20       EC50

Value [mg/l]              5.22       42.41         n.d.

lower 95%-cl            2.18        27.24        n.d.

upper 95%-cl            9.05       72.15         n.d.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs. inhibited growth rate at average exposure concentrations of 9.7 mg/L and higher. Yield was significantly inhibited at average exposure concentrations of 3.4 mg/L and higher.

The EC50 for growth rate inhibition (72h-ERC50) was beyond the range tested, i.e. >99 mg/L.
The 72h-EC10 for growth rate inhibition was 5.2 mg/L (95% confidence interval 2.2 - 9.1 mg/L).
The 72h-NOEC for growth rate inhibition was 3.4 mg/L.

The EC50 for yield inhibition (72h-EYC50) was 14 mg/L with a 95% confidence interval ranging from 9.5 to 23 mg/L.
The 72h-EC10 for yield inhibition was 0.49 mg/L (95% confidence interval 0.14 - 1.1 mg/L).
The 72h-NOEC for yield inhibition was 0.72 mg/L.



Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Benzenesulfonic acid, 4-C15-C16-sec-alkyl derivs..

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the ISO International Standard 8692, 2012.

The batch of Benzenesulfonic acid, 4-C15-C16-sec-alkyl derivs. tested was a dark brown viscous liquid with a purity of 89.2% and completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test. Preparation of test solutions started with a nominal concentration of 100 mg/l. Fifteen minutes of magnetic stirring was applied to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the 100 mg/l concentration in test medium. Test solutions up to and including 32 mg/l were clear and colourless, while the higher test concentration of 100 mg/l was clear and slightly yellow/brown.

Six replicates of exponentially growing algal cultures were exposed to a control, whereas three replicates per concentration were exposed to nominally 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg Benzenesulfonic acid, 4-C15-C16-sec-alkyl derivs. per litre. The initial cell density was 104 cells/ml and the total test period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 of exposure and at the end of the test.

Analysis of the samples taken at the start of the test showed that the actual test concentrations were in agreement with nominal (83-118%). The lowest test concentration decreased below the Limit Of Detection (LOD) within 24 hours of exposure, but was 36-40% of initial at the end of the test. Nominal concentrations of 0.32 and 1.0 mg/l decreased to 53-67% of initial after 24 hours of exposure but were 65-86% of initial at the end of the test. The higher concentrations remained stable (83-102% of initial). Given these results, the effect parameters were based on the average exposure concentrations, i.e. 0.051, 0.18, 0.72, 3.4, 9.7, 33 and 99 mg/l.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Effect parameters based on growth rate:

NOErC 3.4 mg/L

ErC10 5.2 mg/L (95% confidence interval 2.2 -9.1 mg/L)

ErC20 42 mg/L (95% confidence interval 27 -72 mg/L)

ErC50 > 99 mg/L

Effect parameters based on yield:

NOEyC 0.72 mg/L

EyC10 0.49 mg/L (95% confidence interval 0.14 - 1.1 mg/L)

EyC20 1.6 mg/L (95% confidence interval 0.64 -2.8 mg/L)

EyC50 14 mg/L (95% confidence interval 9.5 -23 mg/L)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure following national standard. No analytical verification of the test concentrations.
Qualifier:
according to guideline
Guideline:
other: German industrial standard test guideline DIN 38412, part 9
GLP compliance:
no
Analytical monitoring:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
15.1 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
19.7 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
32.7 mg/L
Basis for effect:
growth rate

Effect values related to nominal concentration of the test substance.

Effect values (endpoint: biomass):
EbC10 (72 h) = 14.9 mg/L
EbC20 (72 h) = 17.9 mg/L
EbC50 (72 h) = 25.4 mg/L

pH:

Concentration

[mg/L]

uninoculated

inoculated

0 h

72 h

72 h

Control

8.2

8.12

9.60

7.812

8.68

8.06

9.44

15.625

8.87

8.09

9.18

31.25

9.21

8.18

8.36

62.5

9.45

8.33

8.38

125

9.69

8.54

8.75

250

9.92

8.80

8.80

500

10.24

9.04

9.03

62.5*

8.25

8.02

8.50

500*

8.08

8.00

8.05

* = neutralised samples

Inhibition may be partly due to the basic pH of the test substance:
- inhibition of the fluorescence after 72 h compared to a control:

Sample

Inhibition [%]

62.5 mg/L

94.8

62.5 mg/L (neutralized)

62.2

500 mg/L

100.0

500 mg/L (neutralized)

94.6

Conclusions:
The 72 hr ErC50 of MIPA to Desmodesmus subspicatus was 32.7 mg/L, while the ErC10 was 15.1 mg/L.

Description of key information

The toxicity of the read across substance Benzenesulfonic acid, 4-C15-16-sec-alkyl derivs. was assessed in a fresh water algal groth inhibition test with Pseudokirchnerella subcapitata according to OECD guideline 201. The ErC10 of 5.2 mg/L (95% CL 2.2 -9.1 mg/L), NOErC of 3.4 mg/L and the ErC50 > 99 mg/L after 72 hours of exposure can be considered reliable without restrictions.

The 72 hr ErC50 of the second read across substance (MIPA) to Desmodesmus subspicatus was 32.7 mg/L while the ErC10 was 15.1 mg/L.

As no consistent pattern of sensitivity was observed between acute and chronic values of the two read across substances, as a precautionary approach the worst case value was used for Benzenesulfonic acid, 4 -C15 -16 -sec-alkyl derivs. MIPA salt.

Key value for chemical safety assessment

EC50 for freshwater algae:
32.7 mg/L
EC10 or NOEC for freshwater algae:
5.2 mg/L

Additional information

The conducted study with Pseudokirchnerella subcapitata according to OECD guideline 201did not produce a defined ErC50 (72h) value, since only less inhibition of the growth rate was observed. The following estimated effect concentrations are based on the measured mean concentrations of the test material.

Effect parameters based on growth rate:

NOErC 3.4 mg/L

ErC10 5.2 mg/L (95% confidence interval 2.2 -9.1 mg/L)

ErC20 42 mg/L (95% confidence interval 27 -72 mg/L)

ErC50 > 99 mg/L

Effect parameters based on yield:

NOEyC 0.72 mg/L

EyC10 0.49 mg/L (95% confidence interval 0.14 - 1.1 mg/L)

EyC20 1.6 mg/L (95% confidence interval 0.64 -2.8 mg/L)

EyC50 14 mg/L (95% confidence interval 9.5 -23 mg/L)