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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:
from 2017-05-29 to 2017-07-12 with the definitive exposure phase from 2017-05-30 to 2017-07-12
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Determination of the test item
All concentration levels and the controls were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Vehicle:
no
Details on test solutions:
Water Accommodated Fraction (WAF)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, complex mixtures and UVCB substances, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure was expressed in terms of the concentration of the test item in water at the start of the mixing period (loading concentration) if the measured concentrations of the main constituents are within ±20 % of the nominal values. Otherwise, the geometric mean measured concentrations of the main constituents were used.

Since the test item is labeled as UVCB but water solubility is above the limit loading concentration, WAF approach is used for media preparation but test media is considered to be a true solution of the test item.

Preparation of the Loading concentrations
Five test item loading concentrations were prepared with a nominal loading of the test item of 6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L (spacing factor 2). The loading concentrations are based on the results of a preliminary range finding test (non-GLP, open system). For each loading concentration an appropriate amount of the test item was weighed. The test item was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water. These dispersions were shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the loading concentrations were removed by siphoning (from the approximate bottom of the glass flask). The resulting test item solutions were used in the test. The loading concentrations were checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative.

Test concentration
In view of substances labeled as UVCB, all terms related to concentration levels should be given as loading levels because partly dissolved compounds and mixtures cannot be related to concentrations. Therefore, the main constituents were analytically monitored and these concentrations are considered as test concentrations.

Control
Six replicates (without test item) were tested under the same test conditions as the test item replicates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
Not specified
Test temperature:
min.: 22.0 max.: 23.5 mean value: 22.8
pH:
Nominal test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
100 7.90 8.26
50.0 7.94 8.35
25.0 8.00 8.75
12.5 8.02 8.94
6.25 7.99 8.96
Control 7.98 9.07
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6839 cells/mL
- Control end cells density: Mean 2103580 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5771 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the saturated solution.

Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: CI: 15.1 - 25.7
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14.8 mg/L
Nominal / measured:
nominal
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):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: The test media clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-04-18 to 2017-04-21. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.559 0.754 ± 0.518
95% confidence interval 0.538 – 0.581
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95% confidence interval 0.242 – 0.341
SD = Standard deviation
Reported statistics and error estimates:
EC-values and statistical analyses
EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.

Cell Densities

Nominal test item Concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

6839

15877

101823

473671

2

6839

15240

112602

504715

3

6839

15245

126881

574647

Mean

6839

15454

113769

517678

 50.0

1

6839

20120

123499

638463

2

6839

21042

163210

688468

3

6839

19506

143594

679456

Mean

6839

20223

143434

668796

 25.0

1

6839

23792

158435

1009070

2

6839

25012

167798

848842

3

6839

19298

170560

993533

Mean

6839

22701

165598

950482

 12.5

1

6839

22119

180918

1067941

2

6839

32468

201951

1519423

3

6839

19450

208774

1195105

Mean

6839

24679

197214

1260823

   6.25

1

6839

25396

200370

1365398

2

6839

25185

216370

1413092

3

6839

28126

190059

1496191

Mean

6839

26236

202266

1424894

Control

1

6839

25779

265913

1738932

2

6839

34762

344107

2078406

3

6839

38829

373894

2338586

4

6839

28462

320255

2341746

5

6839

28690

326493

1993845

6

6839

29867

374585

2129962

Mean

6839

31065

334208

2103580


Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values (with ascorbic acid) are marked (+), not significant differences are marked (-).

 

Nominal test item concentration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.41

26

 

466832

78

2

 

1.43

25

 

497876

76

3

 

1.48

23

 

567808

73

Mean

(+)

1.44

24

(+)

510839

76

 50.0

1

 

1.51

21

 

631624

70

2

 

1.54

19

 

681629

67

3

 

1.53

20

 

672617

68

Mean

(+)

1.53

20

(+)

661957

68

 25.0

1

 

1.67

13

 

1002231

52

2

 

1.61

16

 

842003

60

3

 

1.66

13

 

986694

53

Mean

(+)

1.64

14

(+)

943643

55

 12.5

1

 

1.68

12

 

1061102

49

2

 

1.80

6

 

1512584

28

3

 

1.72

10

 

1188266

43

Mean

(+)

1.74

9

(+)

1253984

40

   6.25

1

 

1.77

7

 

1358559

35

2

 

1.78

7

 

1406253

33

3

 

1.80

6

 

1489352

29

Mean

(+)

1.78

7

(+)

1418055

32

Control

1

 

1.85

 

 

1732093

 

2

 

1.91

 

 

2071567

 

3

 

1.95

 

 

2331747

 

4

 

1.95

 

 

2334907

 

5

 

1.89

 

 

1987006

 

6

 

1.91

 

 

2123123

 

Mean

 

1.91

 

 

2096741

 


Section-by-Section and Average Specific Growth Rates of the Control Group (0 – 72 hours)

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.33

2.33

1.88

1.85

0.504

27.3

23.5

2

1.63

2.29

1.80

1.91

0.346

18.2

3

1.74

2.26

1.83

1.95

0.281

14.5

4

1.43

2.42

1.99

1.95

0.499

25.6

5

1.43

2.43

1.81

1.89

0.504

26.6

6

1.47

2.53

1.74

1.91

0.549

28.7

 

 

 

Mean

1.91

 

 

 

 

 

SD ±

0.04

 

 

 

 

CV [%]

1.95

 

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:
yes
Conclusions:
In this study, Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl dervis., sodium salt was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with
95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L and 20.2 (15.1 – 25.7) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were < 6.25 mg/L, respectively.
Executive summary:

The toxicity of Naphthalenesulfonic acid,bis(1-methylethyl)-, methyl dervis., sodium salt (Batch no.:1452486) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3 from 2017-05-29 to2017-07-12 with the definitive exposure phase from 2017-05-30 to 2017-07-12,at the test facility. The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. Five test item concentrations were prepared with dilution water following protocol for preparation of water accommodated fractions (WAF) with the nominal test item concentrations 6.25 - 12.5 – 25.0 – 50.0 - 100 mg/L (factor 2). For each concentration level an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water. The dispersions were shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the loading concentrations were removed by siphoning (from the approximate centre of the glass flask).

Since the test item is labeled as UVCB but water solubility is above the limit loading concentration, WAF approach is used for media preparation but test media is considered to be a true solution of the test item.

Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The test media were clear throughout the test period. The concentrations of the test itemNaphthalenesulfonicacid,bis(1-methylethyl)-, methyl dervis., sodium saltin all test concentration levels with algae and the control were analytically verified by UPLC-DAD at the start and at the end of exposure.

The measured concentrations of Naphthalenesulfonic acid,bis(1-methylethyl)-, methyl dervis., sodium saltin the fresh media (0 h) were between 103 and 120% of the nominal values. The measured concentrations in the media after 72 hours were between 99 and 119% of the nominal values. The measured test item concentrations were all within ± 20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.

 

Therefore, all effect values given are based on the nominal test item concentrations ofNaphthalenesulfonicacid,bis(1-methylethyl)-, methyl dervis., sodium salt.


 

 

NOEC, LOEC, ECx-values and 95% Confidence Intervals of Naphthalenesulfonicacid,bis(1-methylethyl)-, methyl dervis., sodium salt(0 - 72 hours)

                  based on nominal test item concentrations [mg/L]

 

Growth Rate Inhibition

NOEC

< 6.25

LOEC

6.25

ErC10

14.8 (10.8 – 19.7)

ErC20

50.3 (40.0 – 64.2)

ErC50

> 100

 

Yield Inhibition

NOEC

< 6.25

LOEC

6.25

EyC10

< 6.25

EyC20

< 6.25

EyC50

20.2 (15.1 – 25.7)

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
03-10-2011 to 11-10-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance, including chemical analyses
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See chapter 13 for more details on the Read across
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The NaHCO3 concentration of the test medium was 150 mg/L to maintain a more constant pH during the test. Biomass (dw/mL) was not determined at the end of the test in the control vessels. The chemical analyses were also done on parallel vessels without alg
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
At the start of the test, samples were taken from all test concentrations and the control and stock solution. At the end of the test samples were taken from all parallel concentrations and the parallel control as well as from all actual test vessels at all concentrations. All samples were diluted with leaching solution.

At least 10 mL was sampled in each case this was diluted 1:1 with leaching solution. The samples were then gently shaken and transferred into the analytical vials for analysis. With each transfer step the pipettes used were rinsed with the solution to be analysed and the solution discarded before sampling for the actual analysis.
Samples from the actual test replicates were filtered using a 45 µm filter to remove algae. Filters were primed with the relevant solution before use.
Vehicle:
no
Details on test solutions:
The test substance is dispersible in water. A stock solution of 1.044 g/L of test material was prepared in a 250 mL volumetric flask. An accurately measured amount of 0.2511 g of test material, weighed out on an analytical balance, was loaded in 250 mL test medium. The stock solution was then stirred and a clear homogenous solution was obtained.
The pH of the stock solution was checked and found to be 8.1, therefore no adjustment was necessary.
The stock solution was stirred while adequate amounts were taken for test solutions.

The test solutions were prepared by addition of the adequate amounts of stock solution to the test vessels, then dilution water was added up to the required final volume.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: P. subcapitata
- Strain: (CCAP 278/4)
- Source (laboratory, culture collection): obtained from the Culture Collection of Algae and Protozoa SAMS Research Services Ltd. Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland
- Method of cultivation: ultures on sloped agar tubes were stored at 4°C in the dark until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase.

ACCLIMATION
- Acclimation period: No
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Standard OECD medium with 150 mg/L NaHCO3
Test temperature:
21.3 to 22.0 °C
pH:
8.2 to 9.1
Dissolved oxygen:
Not measured
Salinity:
Standard OECD medium
Nominal and measured concentrations:
Nominal and measured: 10, 30, 90, 270 and 810 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks containing 40 mL of medium. The test flasks were closed with cotton-wool stoppers
- Type (delete if not applicable): open
- Initial cells density: 1*10^4 cells
- Control end cells density: 9.13 * 10^5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes at start of the test
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 108.3 µmol/m2/s and 108.1 µmol/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3
- Range finding study
- Results used to determine the conditions for the definitive study: No inhibition at 10 mg/L and minor inhibition at 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
135 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Active ingredient content is 52.95%
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 810 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
active ingredient content is 52.95%
Basis for effect:
growth rate
Details on results:
CHEMICAL ANALYSIS
The results of measured concentrations in the test series were all above 80% of the nominal concentrations.
At the start of the test the responses seen in the control sample and in 90 mg/L were not as expected. These samples were reanalyzed with the series at the end of the test and were then found to be close to nominal.
The measured concentrations in the parallel series were all between 80 and 120% of the nominal values and in the same range as the samples taken from the test vessels.
Also the stock solutions used to make the test solutions showed a good recovery of 92%. Because the measured concentrations were all close to nominal, nominal test substance concentrations are used for endpoint calculations.
Endpoints are based on test chemical as received not yet corrected for purity.
Results with reference substance (positive control):
found to be between the acceptable EC50 values of 0.25 to 2.0 mg/L.
Reported statistics and error estimates:
The EC10,20,50,80 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance.
The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using threshold values from the William’s test. Confidence limits were computed on the basis of Fieller’s theorem if possible. All computations were performed using the TOXCALC version 5.023 program.
Validity criteria fulfilled:
yes
Conclusions:
The test is reliable because it is performed according to standard OECD guideline 201 under GLP condition and all validity criteria were fulfilled. The test substance was stable during the test and >80% of the nominal test substance concentration was measured at start and end of the test. Therefore nominal test concentrations were used for the dose-response curve. The calculated endpoints are therefore adequate for C&L and risk assessment purposes.
Executive summary:

In order to predict effects of chemicals in an aquatic environment, the toxicity to freshwater algae was assessed. The algal toxicity was determined in the Algal Growth Inhibition Test in accordance with OECD, EEC and ISO test guidelines and with the OECD Principles of Good Laboratory Practice.

Slight modifications to the guideline were applied to ensure good growth and pH control of the cultures.

 

The toxicity of Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated, sodium salts to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours.

No effect was found on growth rate up to 10 mg/L, therefore NOEC is 10 mg/L and LOEC is 30 mg/L. The ErC10and ErC50 are135 and > 810 mg/L, respectively.

The test was conducted in a mineral salts medium in acclimatized illuminated orbital incubator. The maximum variation in pH was 0.8 units.

The test is valid as shown by:

- the increase of the extinction of the control over 72 h by a factor of 91

- the mean coefficient of variation for section-by-section specific growth rates in the control cultures is 32%.

- the coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures is 1.4%       

The results of the chemical analyses show that the concentrations in the test solutions at the start and at the end of the test in the parallel vessels as well as in the test vessels are close to the nominal concentration. All measured concentration stayed within 80 to 120% of the nominal. Therefore, nominal test substance concentrations are used to calculate the effects.

Description of key information

The toxicity of Sodium diisobutylnaphthalenesulphonate to algae is read across from the structurally related substance naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivatives, sodium salts.

In addition algae rangefinding tests have been performed with two batches of Aromatic Hydrocarbons, C10-13, Reaction Products With Branched Nonene Sulphonated, sodium salt to evaluate which of two relatively similar alkylnaphthalene sulphonates is the more toxic substance. The results obtained indicate that there is a slightly higher toxicity observed for the batch containing a higher fraction of long alkyl chain derivative when compared to the batch with a lower content of long alkyl chain derivative.

The lowest ErC50 is observed in the algae test with naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivatives, sodium salts. This result (ErC50>100 mg/L and the ErC10 =14.8 mg/L) will be used as a worst-case for read across for those naphthalenesulphonates for which the algae data are missing.

Key value for chemical safety assessment

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

Additional information

The toxicity of Naphthalenesulfonic acid,bis(1-methylethyl)-, methyl dervis., sodium salt to Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and EC no C.3. 

The study was conducted under static conditions with the nominal test item concentrations 6.25 - 12.5 – 25.0 – 50.0 - 100 mg/L (factor 2). For each concentration level an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water. The dispersions were shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the loading concentrations were removed by siphoning (from the approximate centre of the glass flask).

Since the test item is labeled as UVCB but water solubility is above the limit loading concentration, WAF approach is used for media preparation but test media is considered to be a true solution of the test item.

Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The test media were clear throughout the test period. The concentrations of the test item Naphthalenesulfonicacid,bis(1-methylethyl)-, methyl dervis., sodium salt in all test concentration levels with algae and the control were analytically verified by UPLC-DAD at the start and at the end of exposure.

The measured concentrations of Naphthalenesulfonic acid,bis(1-methylethyl)-, methyl dervis., sodium saltin the fresh media (0 h) were between 103 and 120% of the nominal values. The measured concentrations in the media after 72 hours were between 99 and 119% of the nominal values.The measured test item concentrations were all within ± 20% of the nominal concentrations. This indicates that the test item concentrations were successfully maintained for the duration of the test.

 

Therefore, all effect values given are based on the nominal test item concentrations of Naphthalenesulfonicacid,bis(1-methylethyl)-, methyl dervis., sodium salt.

The observed ErC50 and ErC10 are >100 and 14.8 mg/L respectively.