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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 2017-04-24 to 2017-06-26 with the definitive exposure phase from 2017-06-20 to 2017-06-23,
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 loading 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. Additionally, analysis was done on the stock solution at test start. To evaluate if spiking the test solutions is performed correctly and to evaluate the stability of the test item under test conditions, separate replicates without algae of the two loadings 0.0320 and 0.0800 mg/L were analysed at test start and test end. Adsorption to glass was analytically verified from all concentration levels and the two additional loadings without algae at the end of the exposure. The vessels were rinsed carefully before analysis, to remove adhering algae. The analyzed constituents are considered representative for the exposure to the diamine fraction of the product. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

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, 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 original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fractio
Five water accommodated fractions (WAF) were prepared with an nominal loading of the test item of 0.00513 – 0.0128 – 0.0320 – 0.0800 – 0.200 mg/L (spacing factor 2.5). The loading levels are based on the results of a preliminary range finding test (non-GLP, open system) and the first definitive test.

For a loading of 0.5 mg/L 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 (see Table 2). The dispersion was shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, WAF was removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fraction (WAF) was used as a stock solution. All test loading levels were prepared by diluting the WAF with a loading of 0.5 mg/L.

Test loading
Per definition of the WAF, all terms related to concentration level
were given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

Control
Six replicates (without test item) were tested under the same test conditions as the test item replicates.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 Mean measured pH-values
item loadings initial main
constituents concentration
[mg/L] [mg m.c./L] Start; 0 hours End; 72 hours
0.200 0.0330 7.52 8.03
0.0800 0.0132 7.56 8.34
0.0320 0.00529 7.62 8.94
0.0128 0.00211 7.72 9.10
0.00513 0.000846 7.87 9.20
Control 7.90 9.36
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Nominal and measured concentrations:
Nominal test substance concentrations: 0, 0.00512, 0.0128, 0.032, 0.08 and 0.2 mg/L
Nominal amine fraction concentrations: 0, 0.00267, 0.00668, 0.0167, 0.04176 and 0.1044 mg/L (=52.2% of test substance).
Nominal main constituent concentrations: 0, 0.00235, 0.00587, 0.0147, 0.0367 and 0.0917 mg/L (main constituents C18:1 diamine, C16 diamine and C18 diamine represent 87.8% of amine fraction in the test item)
Measured main constituent concentrations: 0, 0.000846, 0.00211, 0.00529, 0.0132 and 0.0330 mg/L (Geometric mean measured concentrations of the main constituents)
Calculated concentration of the amine fraction in the test item: 0, 0.00964, 0.00240, 0.00603, 0.0150 and 0.0376 mg/L (calculated from geometric mean by multiplying by 1/0.878)
Calculated concentration of the test item: 0, 0.00185, 0.00460, 0.01154, 0.0288 and 0.072 mg/L (calculated from geometric mean of the amine fraction and multiplying by 1/0.522)
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 2025631 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 0.064
Na2EDTA 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: 5878 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 definitive test.
Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.055 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: Calculated test item concentration using the mean measured initial concentrations of the main constituents
Basis for effect:
growth rate
Remarks on result:
other: Cl: 0.0415 - 0.0659
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.019 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: Calculated test item concentration using the mean measured initial concentrations of the main constituents
Basis for effect:
growth rate
Remarks on result:
other: Cl: 0.0101 - 0.0255
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
0.005 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
0.154 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 0.115-0.183
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.001 mg/L
Nominal / measured:
not specified
Conc. based on:
other: calculated main constituents
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.01 mg/L
Nominal / measured:
not specified
Conc. based on:
other: calculated main constituents
Basis for effect:
other: yield
Remarks on result:
other: CI: 0.00836 - 0.0111
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
0.058 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: CI: 0.0506 - 0.0671
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.025 mg/L
Nominal / measured:
not specified
Conc. based on:
other: calculated main constituents
Basis for effect:
growth rate
Remarks on result:
other: CI: 0.0190 - 0.0302
Details on results:
The use of the mean measured initial concentration is considered justified because the substance sorbs strongly to algae and the growth rate . Due to that nigligible concentrations were observed in the aqueous phase after 72h and because sorption to glassware is observed to be nigligible. This approach is in agreement with point 40 of the OECD 201 guideline.

- 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:
EL/C-values and statistical analyses
EL/C10-, EL/C20- and EL/C50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEL/NOEC, LOEL/NOEC and statistical analyses
The NOEL/NOEC and LOEL/LOEC were determined by calculation of statistically significant differences of growth rates and yield. The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was calculated with a significance level of 0.01.
Levene’s test on variance homogeneity was calculated with a significance level of 0.01.
Dunnett’s multiple -t-test was calculated with a significance level of 0.05 for yield.
Monotonicity of Concentration/Response was calculated by non-parametric trend analysis by contrasts (significance level 0.05) for growth rate.
SD Jonckheere-Terpsta test procedure rate was done for growth with a significance level of 0.05 for growth rate.

Calculation of initial main constituents concentrations
The recoveries for the lowest two loadings were < LOQ at test start. Therefore, and as the recoveries in all loadings > LOQ were very similar, a mean value of recovery percentages of the upper three loadings containing algae was calculated, which was 36% of the nominal values. This percentage was applied for all loadings to calculate the expected recovery. This is considered to reflect the actual concentrations in the best possible and reliable manner.

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.
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.
•ToxRat Version 3.2.1, ToxRat Solutions GmbH

Cell Densities

Nominal test item loading

Calculated initial main constituents concentration

Replicate

Cell density [cells/mL]

[mg/L]

[mg m.c./L]

No.

0 hours

24 hours

48 hours

72 hours

0.200

0.0330

1

6839

7412

6052

17457

2

6839

6994

13077

38384

3

6839

8960

13279

55978

Mean

6839

7789

10803

37273

0.0800

0.0132

1

6839

17288

59952

501994

2

6839

17855

93320

817359

3

6839

18467

94520

841147

Mean

6839

17870

82597

720167

0.0320

0.00529

1

6839

20694

152940

1588038

2

6839

19889

171865

1416574

3

6839

21323

169665

1415404

Mean

6839

20635

164823

1473339

0.0128

0.00211

1

6839

24219

199978

1871216

2

6839

25109

207165

1680441

3

6839

23309

199335

1713036

Mean

6839

24212

202159

1754898

0.00513

0.000846

1

6839

22590

199873

1972924

2

6839

24214

212619

1846169

3

6839

26467

196514

1770035

Mean

6839

24424

203002

1863043

Control

1

6839

27131

227565

1966779

2

6839

28076

218904

2267338

3

6839

25756

236858

1786947

4

6839

26276

244220

2072993

5

6839

28711

252295

1996800

6

6839

25390

243236

2062927

Mean

6839

26890

237180

2025631


Evaluation after 72 hours

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

Nominal test item loading

Calculated initial main constituents concentration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

[mg m.c./L]

No.

[d-1]

[%]

[cells/mL]

[%]

0.200

0.0330

1

 

0.312

84

 

10618

99

2

 

0.575

70

 

31545

98

3

 

0.701

63

 

49139

98

Mean

(+)

0.529

72

(+)

30434

98

0.0800

0.0132

1

 

1.43

24

 

495155

75

2

 

1.59

16

 

810520

60

3

 

1.60

15

 

834308

59

Mean

(+)

1.54

19

(+)

713328

65

0.0320

0.00529

1

 

1.82

4

 

1581199

22

2

 

1.78

6

 

1409735

30

3

 

1.78

6

 

1408565

30

Mean

(+)

1.79

6

(+)

1466500

27

0.0128

0.00211

1

 

1.87

1

 

1864377

8

2

 

1.84

3

 

1673602

17

3

 

1.84

3

 

1706197

15

Mean

(+)

1.85

3

(+)

1748059

13

0.00513

0.000846

1

 

1.89

0

 

1966085

3

2

 

1.87

2

 

1839330

9

3

 

1.85

2

 

1763196

13

Mean

(-)

1.87

1

(-)

1856204

8

Control

1

 

1.89

 

 

1959940

 

2

 

1.94

 

 

2260499

 

3

 

1.86

 

 

1780108

 

4

 

1.91

 

 

2066154

 

5

 

1.89

 

 

1989961

 

6

 

1.90

 

 

2056088

 

Mean

 

1.90

 

 

2018792

 


 

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

1.38

2.13

2.16

1.89

0.441

23.4

24.5

2

1.41

2.05

2.34

1.94

0.474

24.5

3

1.33

2.22

2.02

1.86

0.469

25.3

4

1.35

2.23

2.14

1.91

0.486

25.5

5

1.44

2.17

2.07

1.89

0.400

21.1

6

1.31

2.26

2.14

1.90

0.516

27.1

 

 

 

Mean

1.90

 

 

 

 

 

SD ±

0.03

 

 

 

 

CV [%]

1.37

 

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:
yes
Conclusions:
In this study, the test item 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 loadings: The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 0.154 (0.115 – 0.183) mg/L and 0.0583 (0.0506 – 0.0671) mg/L, respectively. The EL10-values with 95% confidence intervals for inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were 0.0524 (0.0281 – 0.0710) and 0.0836 (< 0.00513 – 0.0161) mg/L, respectively.
The following effect values are based on the calculated initial main constituents concentrations: The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0254 (0.0190 – 0.0302) and 0.00962 (< 0.000846 – 0.00266) mg m.c./L, respectively. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.00863 (0.00462 – 0.0117) and 0.00138 (0.00836 – 0.0111) mg m.c./L, respectively.
The following effect values are based on the calculated initial test item concentrations which were calculated from the observed effect values for the main constituents: The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0554 (0.0415 – 0.0659) mg m.c./L and 0.02099 (< 0.00185 – 0.00580) mg test item/L, respectively. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.0188 (0.0101 – 0.0255) and 0.00301 (0.00182 – 0.0242) mg test item/L, respectively.
Executive summary:

The toxicity of the test item 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 2017-04-24 to 2017 -06-26 with the definitive exposure phase from 2017-06-20 to 2017-06-23, at the test facility. The aim of the study was the determination of NOEL, LOEL, EL10, EL20- and EL50-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. As the test item is poorly water soluble and an UVCB substance, five water accommodated fractions (WAF) with the nominal test item loadings 0.00513 -0.0128 – 0.0320 - 0.0800 - 0.200 mg/L (factor 2.5), corresponding to the calculated initial main constituents concentrations 0.000846 – 0.00211 – 0.00529 – 0.0132 – 0.0330 mg m.c./L were prepared with dilution water. For the loading of 0.500 mg/L 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 into a brown glass flask with an appropriate amount of dilution water. The dispersion was shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the WAF was removed by siphoning (from the approximate centre of the glass flask). The resulting WAF was used as a stock solution and all tested loading levels were prepared by diluting the WAF, since the concentrations were too low for direct weighing.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 Redicote 814M in all test concentration levels with and without algae and the control were analytically verified by LC-MS/MS at the start and at the end of exposure. Additionally, the adsorption to glass was analytically verified from all concentration levels with and without algae.

 

The measured concentrations of the test item in the fresh media (0 h) with and without algae were between < LOQ and 55% of the nominal concentrations for the three main constituents C16 diamine, C18:1 diamine and C18:0 diamine. The measured concentrations in the media after 72 hours with and without algae were between < LOQ and 7% of the nominal concentrations for the three main constituents C16 diamine, C18:1 diamine and C18:0 diamine. The measured concentrations of the adsorption to glass at the end of the exposure with and without algae were between < LOQ and 13% of the nominal values for the component C16 diamine, < LOQ for the component C18:1 diamine and < LOQ and 17% for the component C18:0 diamine.

 

Therefore, all effect values given are based on the nominal test item loadings and the calculated initial main constituents concentration of the test item. In addition were the main constituent concentrations recalculated to the test item concentrations using the fraction of different constituents in the test item.


 

NOEL/NOEC, LOEL/LOEC, ELx/ECx-values and 95% Confidence Intervals of Redicote 814M (0 - 72 hours)

                  based on nominal test item loadings and the calculated initial main constituents and calculated test item concentrations [mg/L]

 

Growth Rate Inhibition

Nominal test item loading [mg/L]

Calculated main constituents
[mg m.c./L]

Calculated test item
[mg./L]

NOEL/NOEC

0.00513

0.000846

0.00185

LOEL/LOEC

0.0128

0.00211

0.00460

ErL10/ ErC10

0.0524 (0.0281 – 0.0710)

0.00863 (0.00462 – 0.0117)

0.0188 (0.0101 – 0.0255)

ErL20/ ErC20

0.0834 (0.0665 – 0.139)

0.0138 (0.0110 – 0.0230)

0.0301 (0.0240 – 0.0502)

ErL50/ ErC50

0.154 (0.115 – 0.183)

0.0254 (0.0190 – 0.0302)

0.0554 (0.0415 – 0.0659)

 

Yield Inhibition

Nominal test item loading [mg/L]

Calculated main constituents [mg m.c./L]

Calculated test item [mg/L]

NOEL/NOEC

0.00513

0.000846

0.00185

LOEL/LOEC

0.0128

0.00211

0.00460

ErL10/ ErC10

0.00836 (< 0.00513 – 0.0161)

0.00138 (0.000836 – 0.0111)

0.00301 (0.00182 – 0.0242)

ErL20/ ErC20

0.0229 (0.0168 – 0.0304)

0.00379 (0.00278 – 0.00503)

0.00827 (0.00607 – 0.01100)

ErL50/ ErC50

0.0583 (0.0506 – 0.0671)

0.00962 (< 0.000846 – 0.00266)

0.02099 (0.00185 – 0.00580)
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008-02-04 to 2008-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Complete report, test performed under GLP, all validity criteria fulfilled
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 9-20 °C
- Boiling point: > 250 °C
- Vapour pressure: < 1 hPa (20 °C)
- Water solubility (under test conditions): Insoluble
- Henry's law constant: Not applicable
- log Pow: Not applicable
- pKa: Not applicable
- Stability in water: Not applicable
- Stability in light: Not applicable
- pH dependance on stability: Not applicable

OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: Not applicable
- Other:
- Density: 840 kg/m3 (25 °C)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 10.0 - 32.0 - 100 - 320 - 1000 µg/L
- Sampling method: Each sample was diluted 1:2 with 2-propanol containing 4 % formic acid directly after sampling for stabilisation
- Sample storage conditions before analysis: Room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 20 mg/L, freshly prepared with dilution water 30 min before application, sonificated for 30 min at 40 °C
- Differential loading: 10-32-100-320-1000 µg/L (factor: √10)
- Controls: six replicates without test item, tested under the same conditions as the test replicates
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not observed
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE•m-2•s-1 for 24 h per day

ACCLIMATION
- Culturing media and conditions (same as test or not): Inoculum: Nutrient medium Z according to Luettge (1994), conditions same as for test; test replicates: natural river water, other conditions same as for test
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
After 72 h algae were transferred from nominal concentrations 320 – 1000 µg/L and control into fresh untreated medium (0.5 – 1 mL algae suspension from each replicate were filled up with dilution water to 10 mL) and allowed to grow for further 5 d to determine whether the effect of the item is reversible.
Hardness:
Not measured
Test temperature:
Mean: 21.5 °C, Min: 21 °C, Max: 22 °C
pH:
Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
1000 7.76 7.70
320 7.77 7.66
100 7.73 7.87
32 7.76 8.04
10 7.86 7.76
Control 7.92 8.03
Dissolved oxygen:
Not measured
Salinity:
Not measured
Nominal and measured concentrations:
Nominal test item concentrations: 10-32-100-320-1000 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- 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 approximately 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 4971 cells/mL
- Control end cells density: Mean 774306 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water, river grane, located in the low mountain range "Harz", D-38685 Langelsheim, Herzog-Julius-Hütte, Im Granetal; additionally 50 % of the components of the dilution water acc. to the guideline were added to enable a sufficient growth of algae
- Particulate matter: 2.5 mg/L
- DOC 1.5 mg C/L
- Conductivity: 149 µS (natural river water)
- Salinity: Not measured
- Culture medium different from test medium: Nutrient medium Z according to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: 24 h
- Light intensity and quality: 60-120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Microscopic
- Chlorophyll measurement: fluorimeter; Chlorophyll-a-fluorescence, excitation at 435 nm, emission at 685 nm; measurements at test start and every 24 h, each replicate was measured 6-fold, value (flash) no. 3 - 6 were used for calculations
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Range finding study
- Test concentrations: 10-100-1000 µg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
507 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 468 - 543
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
188 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 167-206
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: none
- Colour differences: none
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: at test end of the concentration levels 320 - 1000 µg/L
- Other: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None

The study meets the validity criteria of the guideline:
- The cell growth increased 156-fold after 72 h in the control (required: 16-fold).
- The temperature during the test was in the range of 21-24 °C. The difference from the mean was within ± 2 °C: min.: 21 °C, max.: 22 °C mean value: 21.5 °C.
- The pH-value in the control replicates increased not higher than 1.5 units: from 7.92 to 8.03.
- The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was 27.71 % (required: < 35 %).
- Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1.94 % (required: < 7 %).
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 1.01 (CI 0.90 - 1.13) mg/L
Yield inhibition: 0.42 (CI 0.37 - 0.48) mg/L
Reported statistics and error estimates:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using maximum likelihood analyses.

Table 1: EC10-, EC20- and EC50- values (0-72 h) of N-Oleyl-1,3-diaminopropane

                based on nominal test item concentrations [µg/L]

                            

Inhibition of yield

EyC10 (95 % confidence interval)

113 (98 – 131)

EyC20 (95 % confidence interval)

137 (121 – 158)

EyC50 (95 % confidence interval)

214 (186 – 243)

Rate-related inhibition

ErC10 (95 % confidence interval)

188 (167 – 206)

ErC20 (95 % confidence interval)

267 (244 - 291)

ErC50 (95 % confidence interval)

507 (468 – 543)

Table 2:     Cell Densities

Nominal
test item concentration

Replicate

Cell density [cells/mL]

[µg/L]

No.

0 h

24 h

48 h

72 h

 

1000

1

4971

10547

7050

12720

 

2

4971

9255

8373

13602

 

3

4971

11649

9948

9255

 

Mean

4971

10484

8457

11859

 

320

1

4971

38393

108669

207390

 

2

4971

33920

100605

159573

 

3

4971

39653

142595

230732

 

Mean

4971

37322

117290

199232

 

100

1

4971

42519

223266

667416

 

2

4971

43275

210351

685056

 

3

4971

41291

215076

808536

 

Mean

4971

42362

216231

720336

 

32

1

4971

41984

239961

892326

 

2

4971

41417

260751

868071

 

3

4971

45858

219171

848856

 

Mean

4971

43086

239961

869751

 

10

1

4971

39716

237126

789006

 

2

4971

44945

288156

777981

 

3

4971

42992

198066

896421

 

Mean

4971

42551

241116

821136

 

Control

1

4971

40661

240276

730101

 

2

4971

44756

225471

674346

 

3

4971

42897

241536

798456

 

4

4971

38298

218226

794361

 

5

4971

41921

201216

748056

 

6

4971

44409

252246

900516

 

Mean

4971

42157

229829

774306

 

Table 3     Evaluation after 72 h

        

Nominal
test item concentration

Replicate

Yield

Inhibition of yield

Growth rate

Rate-related inhibition

[µg/L]

No.

[%]

[d-1]

[%]

1000

1

7749

98.99

0.313

81.37

2

8631

98.88

0.336

80.04

3

4284

99.44

0.207

87.68

Mean

6888

99.10

0.285

83.03

320

1

202419

73.69

1.244

26.04

2

154602

79.90

1.156

31.23

3

225761

70.66

1.279

23.92

Mean

194261

74.75

1.226

27.06

100

1

662445

13.89

1.633

2.86

2

680085

11.60

1.642

2.35

3

803565

-4.45

1.697

-0.94

Mean

715365

7.01

1.657

1.42

32

1

887355

-15.34

1.730

-2.89

2

863100

-12.19

1.721

-2.35

3

843885

-9.69

1.713

-1.90

Mean

864780

-12.41

1.721

-2.38

10

1

784035

-1.91

1.689

-0.45

2

773010

-0.48

1.684

-0.17

3

891450

-15.87

1.732

-2.98

Mean

816165

-6.09

1.702

-1.20

Control

1

725130

1.663

2

669375

1.637

3

793485

1.693

4

789390

1.691

5

743085

1.671

6

895545

1.733

Mean

769335

1.681

Negative values = increase of growth

Table 4:     Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 h)

SD

±

VC %

Mean

VC %

section-by-section                           

0 - 24 h

24 - 48 h

48 - 72 h

Control

1

2.102

1.777

1.111

1.663

0.505

30.35

27.71

2

2.198

1.617

1.096

1.637

0.551

33.68

3

2.155

1.728

1.196

1.693

0.481

28.39

4

2.042

1.740

1.292

1.691

0.377

22.31

5

2.132

1.569

1.313

1.671

0.419

25.08

6

2.190

1.737

1.273

1.733

0.459

26.46

Mean

1.681

 

SD ±

0.03

 

VC %

1.94

 

SD = Standard deviation        VC %= percent coefficient of variation

Table 5:     Recovery of Alga Cells

Nominal test item concentration                   [µg/L]

Incubation time                    [d]

Cell density [cells/mL]

Growth rate after incubation time [d-1]

Initial#

Final

1000

5

1186

72255

0.82

76917

0.83

54867

0.77

320

19923

242229

0.50

203295

0.46

217407

0.48

Control

38715

201909

0.33

250041

0.37
#) calculated from the cell density of the definitive test after 72 h
Validity criteria fulfilled:
yes
Remarks:
The study meets the validity criteria of the guideline
Conclusions:
The test is considered valid as all validity criteria have been fulfilled; Based on the test setup (GLP, Guideline study) and the extensive effort which has been paid to ascertain a realistic exposure of the test organisms and to quantify the extent of this exposure the results are considered to be reliable. Finally, the test result is considered adequate for the evalution of the environmental risks in the EU applying the bulk approach considering the realistic worst case (low) concentration of suspended matter (2.5 mg/L) and DOC (1.5 mg C/L) in the river water used.

Executive summary:

The toxicity of N-Oleyl-1,3-diaminopropane (CAS No. 7173-62-8) (Batch no.: S000902)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from February 04 to 12, 2008. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

An individual test design was applied. Natural river water was used as dilution medium.

The study was conducted under static conditions with an initial cell density of nominal 2 to 5 x 103cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of √10, nominal: 10 - 32 - 100 - 320 - 1000 µg/L. Three replicates were tested for the test item concentrations and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of C18 : 1 diamine, main component of The test substance were analysed at all concentration levels at test start and test end via LC-MS/MS analysis. The measured concentrations at test start were in the range of 91 to 98 % of the nominal values. At the end of the test N-Oleyl-1,3-diaminopropane gave recoveries below the limit of quantification up to 12 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, low DOC and suspended matter content and also because the river water was frozen before use to minimize the microbial activity. This is supported by the monitoring results from the chronic daphnia tests with this test item. In this test only a limited decrease of the test substance was observed over 48 hours. This decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. The larger decrease of the test item during the algae test is therefore most likely caused by sorption of the test substance to algae. These points support the conclusion that the test organisms were fully exposed to the test substance during the test. At the end of the test N-Oleyl-1,3-diaminopropane gave recoveries ranging from LOQ (2.04 µg/L) to 12 % of the nominal values. Therefore all effect values are given based on nominal concentrations of the test item. In this study N-Oleyl-1,3-diaminopropane was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal concentrations): The EC10-values with 95 % confidence intervals for inhibition of yield (EyC10) and specific growth rate (ErC10) after 72 h were 113 (98 to 131) and 188 (167 to 206) µg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of yield (EyC50) and specific growth rate (ErC50) after 72 h were 214 (186 to 243) and 507 (468 to 543) µg/L, respectively.

After 72 h algae were transferred from the nominal concentrations 320 to 1000 µg/L and control to fresh untreated medium and allowed to grow for further 5 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 1000 µg/L (highest test concentration)

Description of key information

One toxicity test with the green alga Pseudokirchneriella subcapitata according to OECD 201 has been performed with"Reaction mass of Amines, N-tallow alkyltrimethylenedi-, (2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3 -diaminopropane ditallate" (R814M). This substance is the salt of tallow-1,3 -diaminopropane (CAS no 1219010 -04 -4) and a mixture of talloil fatty acids, 2 -ethylhexoic and acetic acid. The 72h ErC50 and ErC10 based on main constituents is respectively 0.0254 (95% CL: 0.0190 – 0.0302) and 0.00863 (95% CL: 0.00462 – 0.0117 mg m.c./L.

Finally these results were recalculated to the effect data for the test item resulting in a 72h ErC50 and ErC10 of respectively 0.0554 (95% CL: 0.0430 – 0.0511) and 0.0188 (95% CL: 0.0101 – 0.0255) mg test item/L.

Under aquatic environmental conditions a large extend of the salt is expected to be dissociated and ecotoxicity observed will originate mainly from tallow-1,3 -diaminopropane. No effects (NOEL>=100 mg/L) were observed for the WAF of tall oil fatty acid to algae and daphnids and for 2 -ethyl hexanoic acid the lowest NOEC (21d daphnia) was 25 mg/L (ECHA). Therefore available ecotoxicity data on alkyl-1,3-diamines were included in the dataset. The ecotoxicity of tallow-1,3 -diaminopropane is expected to be reduced when forming a salt with talloil fatty acids or 2 -ethylhexoic acid.

One study with oleyl-1,3 -diaminopropane which was performed in river water which resulted in an ErC10 and ErC50 of respectively 0.188 and 0.507 mg/L.

Recalculating these results to R814M using the tallow-1,3 -diaminopropane content of 52.2% in R814M gives a ErC10 and ErC50 of respectively 0.360 and 0.97 mg/L. These ErC values of 0.360 and 0.97 mg/L will be as a worst-case be used as Key value for risk assessment.

The result as observed in the algae study of 0.0554 mg/L with R814M can be used as Key study for C&L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.97 mg/L
EC10 or NOEC for freshwater algae:
360 µg/L

Additional information

One toxicity test with the green alga Pseudokirchneriella subcapitata according to OECD 201 has been performed with"Reaction mass of Amines, N-tallow alkyltrimethylenedi-, (2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3 -diaminopropane ditallate" (R814M). This substance is the salt of tallow-1,3 -diaminopropane (CAS no 1219010 -04 -4) and a mixture of talloil fatty acids, 2 -ethylhexoic and acetic acid.

Due to the complex structure of the test item, the WAF approach was used. The study was conducted under static conditions over a period of 72 hours using a stock solution which was prepared as a WAF (0.5 mg/L) of the test item. The test solutions were prepared by diluting the WAF to the following nominal concentration range : 0.00513 -0.0128 – 0.0320 - 0.0800 - 0.200 mg/L (factor 2.5). The concentrations of the main amine constituents (diamine C18:1, diamine C16 and diamine C18) of the test item were analytically verified via LC-MS/MS in fresh media at the start of the exposure and in the old media (72h) in all concentration levels and the control. Additionally, the adsorption to glass was analytically verified from all concentration levels with and without algae. The chemical analysis was focused on the amine fraction in the test solutions because this fraction is considered to be the most toxic fraction in the test item. The measured concentrations of the three main constituents (m.c.) of the amine fraction of the test item did not remain within ± 20% of the nominal concentrations and therefore the mean measured initial concentrations of the sum of the three main constituents was calculated and are reported in addition to the nominal loading levels. The use of the mean measured initial concentration is considered justified because of the strong sorption of the test item to the algae (see OECD 201 point 40). The observed mean measured initial concentrations of the main constituents were: 0.000846 – 0.00211 – 0.00529 – 0.0132 – 0.0330 mg m.c./L

The dose effect data derived using the nominal loading concentrations resulted in a 72h ErL50 and ErL10 of respectively 0.154 (95% CL: 0.115 – 0.183) and 0.0524 (95% CL: 0.0281 – 0.0710) mg/L.

The 72h ErC50 and ErC50 based on main constituents is respectively 0.0254 (95% CL: 0.0190 – 0.0302) and 0.00863 (95% CL: 0.00462 – 0.0117 mg m.c./L

Finally these results were recalculated to the effect data for the test item resulting in a 72h ErC50 and ErC10 of respectively 0.0554 (95% CL: 0.0430 – 0.0511) and 0.0188 (95% CL: 0.0101 – 0.0255) mg test item/L.

Under environmental conditions a large extend of the salt is expected to be dissociated and ecotoxicity observed will originate mainly from tallow-1,3 -diaminopropane. Therefore available ecotoxicity data on alkyl-1,3-diamines were included in the dataset. The ecotoxicity of tallow-1,3 -diaminopropane is expected to be reduced when forming a salt with talloil fatty acids or 2 -ethylhexoic acidbutbecause of the availability of real measured acute toxicity data for the test substance (R814M). The availabledataon alkyl-1,3 -diaminopropanes was included for comparison reasons.

For alkyl-1,3 -diaminopropanes two studies with algae are available. One older study from 2000 where a 72h EC50 of 0.046 mg/L for tallow-1,3 -diaminopropane was observed using lab water with a reliability score of 2 e.g. because the exposure concentrations were not quantified and the mean coefficient of variation for section-by-section specific growth rates in the control cultures exceeded 35%. One study with oleyl-1,3 -diaminopropane which was performed in river water which resulted in an ErC10 and ErC50 of respectively 0.188 and 0.507 mg/L.

Recalculating these results to R814M using the tallow-1,3 -diaminopropane content of 52.2% in R814M gives a ErC10 and ErC50 of respectively 0.360 and 0.97 mg/L. These ErC values of 0.360 and 0.97 mg/L will be as a worst-case be used as Key value for risk assessment.

The result as observed in the algae study of 0.0554 mg/L with R814M can be used as Key study for C&L.

There is also an algae study (OECD 201, river water, reliability 1) available with tallow-1,3 -diaminopropane oleate (CAS no 1307863 -78 -0) which can be considered to be a comparable substance to the talloil fatty acid salt of tallow-1,3 -diaminopropane. This study resulted in a 72h ErC10 and ErC50 of respectively 0.324 and 0.509 mg/L.