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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-05-26 to 2009-07-03
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
(Z)-N-(3-aminopropyl)-N'-9-octadecenylpropane-1,3-diamine
EC Number:
249-276-6
EC Name:
(Z)-N-(3-aminopropyl)-N'-9-octadecenylpropane-1,3-diamine
Cas Number:
28872-01-7
IUPAC Name:
N-(3-aminopropyl)-N'-octadec-9-en-1-ylpropane-1,3-diamine
Details on test material:
- Name of test material (as cited in study report): Oleyl dipropylene triamine (CAS No. 28872-01-7)
- Substance type: Surfactant
- Physical state: Paste, pale yellow
- Composition of test material, percentage of components: Primary fatty amine: 4.1 (area %), Di amine: 27.6 (area %), Tri amine: 65.1 (area %), Tetra amine: 3.2 (area %)
- Purity test date: 2009-02-05
- Lot/batch No.: S001256
- Expiration date of the lot/batch: 2017-05-24
- Storage condition of test material: Room temperature, protected from moisture and light, under nitrogen gas

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: From the series of concentrations the second lowest and the second highest test concentration (0.32 and 3.20 mg/L) and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates for the test item analysis after 0 and 72 h were prepared without algae. Analysis after 72 h of these concentrations was carried out to demonstrate stability of the test item. Sorption to the walls of the glass container was checked out of one test replicate (prepared with algae) of a concentration level close to the EC50-value (the second lowest test concentration of 0.32 mg/L).
- Sample storage conditions before analysis: All samples were stored at room temperature, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (10 mg/L) was treated with ultrasound for 15 min at 40 °C. An equilibration phase for 1 h before application was carried out.
- Eluate: Natrual river water
- Differential loading: 0.10 - 0.32 - 1.00 - 3.20 - 10-0 mg/L
- Controls: Six replicates wihtout test item were tested under the same test conditions as the test replicates.

Test organisms

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, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
After 72 h of exposure, 0.5 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from test concentrations 1.0 – 10.0 mg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 – 7 days 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 10.0 mg/L.
Recovery of Alga Cells

Nominal test item concentration [mg/L] Incubation time [d] Cell density [cells/mL] Growth rate after incubation time [d-1]
Initial# Final
10.0 7 3 1881 0.91
3952 1.01
8781 1.13
3.20 18 40558 1.10
4795 0.79
12226 0.93
1.00 3 10956 415223 1.21
299421 1.10
308339 1.11
Control 51801 1599371 1.14
1576467 1.14

Test conditions

Hardness:
Not measured
Test temperature:
Mean: 23 °C, Min: 22 °C, Max: 24 °C
pH:
Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
10.0 8.05 8.10
3.20 8.04 8.01
1.00 8.02 8.04
0.32 8.03 8.10
0.10 8.02 8.09
Control 7.91 7.87
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- 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: 4996 cells/mL
- Control end cells density: Mean 1036016 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



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water of the river Innerste was used. This river is located in
Sarstedt / Ruthe. Water parameters are given in Table 1. Additionally 50 % of the components concentrations of the dilution water (total application volume 6.5 mL/L) acc. to the guideline were added to enable a sufficient growth of algae
Characterisation Data of Natural River Water
River Innerste
Location D-31157 Sarstedt / Ruthe, Ruther Straße, Innerste
Sampling Date 2009-02-12
Weather on Day of Sampling dry, sunny, ca. 1 °C
Colour Slightly, turbid, yellowish
pH 8.08
Conductivity [µS/cm] 887
DOC [mg C/L] 2.8
TOC [mg C/L] 2.9
Ammonium-N [mg N/L] 0.163
Nitrate-N [mg N/L] 6.10
o-Phosphate-P [mg P/L] 0.103
Total Phosphate [mg P/L] 0.152
Suspended Matter [mg/L] 24
Total Hardness [mg CO3/L] 338



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: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 h)

Nominal
Test Item Concentration [mg/L] Rate related Inhibition
[%] Yield Inhibition

[%]
10.0 100 100
1.00 15 59
0.10 0 -1
0.01 -1 -3




Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.518 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 0.348 - 0.594
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.09 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 1.02 - 1.14
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.356 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: (0.314-0.398)
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: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.320 (CI 0.294 - 0.358) 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 standard procedures.

Any other information on results incl. tables

EC10-, EC20-and EC50- values and 95 % Confidence Intervals of
Oleyl dipropylene triamine (CAS No. 28872-01-7) (0-72 h)

                                based on nominal test item concentrations [mg/L]

                               

Rate-related inhibition

ErC10

0.518 (0.348 – 0.594)

ErC20

0.728 (0.674 – 0.776)

ErC50

1.09 (1.02 – 1.14)

Inhibition of yield

EyC10

0.118 ( 0.10 – 0.155)

EyC20

0.181 (0.148 – 0.217)

EyC50

0.356 (0.314 – 0.398)

Cell Densities

 

Nominal
test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 h

24 h

48 h

72 h

 

10

1

4996

0

0

0

 

2

4996

37

0

0

 

3

4996

365

0

99

 

Mean

4996

134

0

33

 

3.20

1

4996

5306

0

0

 

2

4996

6011

1011

0

 

3

4996

7026

1170

553

 

Mean

4996

6114

727

184

 

1.00

1

4996

20525

69569

117019

 

2

4996

17899

65256

96756

 

3

4996

22581

69439

114898

 

Mean

4996

20335

68088

109558

 

0.32

1

4996

28617

159856

563210

 

2

4996

31168

143130

544471

 

3

4996

30859

155446

621879

 

Mean

4996

30215

152811

576520

 

0.10

1

4996

27758

231311

834453

 

2

4996

24487

196410

1004975

 

3

4996

30835

201006

1030083

 

Mean

4996

27693

209576

956504

 

0

(Control)

1

4996

26614

210558

1054429

 

2

4996

28257

211573

982343

 

3

4996

31891

208980

1017811

 

4

4996

30401

222007

1086460

 

5

4996

28593

213080

1028917

 

6

4996

23782

184790

1046137

 

Mean

4996

28256

208498

1036016

 

Evaluation after 72 h

             

Nominal
test item concentration

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

10

1

n.a.

100.00

-4996

100.00

2

n.a.

100.00

-4996

100.00

3

-1.307

100.00

-4897

100.00

Mean

n.a.

100.00

-4963

100.00

3.20

1

n.a.

100.00

-4996

100.00

2

n.a.

100.00

-4996

100.00

3

-0.734

100.00

-4443

100.00

Mean

n.a.

100.00

-4812

100.00

1.00

1

1.051

40.88

112023

89.13

2

0.988

44.44

91760

91.10

3

1.045

41.22

109902

89.34

Mean

1.028

42.18

104562

89.86

0.32

1

1.575

11.42

558214

45.86

2

1.564

12.05

539475

47.68

3

1.608

9.56

616883

40.17

Mean

1.582

11.01

571524

44.57

0.10

1

1.706

4.05

829457

19.55

2

1.768

0.56

999979

3.01

3

1.776

0.10

1025087

0.58

Mean

1.750

1.57

951508

7.71

0

(Control)

1

1.784

1049433

2

1.760

977347

3

1.772

1012815

4

1.794

1081464

5

1.776

1023921

6

1.781

1041141

Mean

1.778

1031020

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

1.673

2.068

1.611

1.784

0.2481

13.9

12.4

2

1.733

2.013

1.535

1.760

0.2401

13.6

3

1.854

1.880

1.583

1.772

0.1643

9.27

4

1.806

1.988

1.588

1.794

0.2004

11.2

5

1.745

2.009

1.575

1.776

0.2186

12.3

6

1.560

2.050

1.734

1.781

0.2485

13.9

Mean

1.778

 

SD ±

0.01

 

VC %

0.64

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Oleyl dipropylene triamine (CAS No. 28872-01-7) 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 specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.518 (0.348 – 0.594) and 0.118 (< 0.10 – 0.155) mg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 1.09 (1.02 – 1.14) and 0.356 (0.314 – 0.398) mg/L, respectively.
The test effect was observed to be reversible at all tested concentrations following 72 h exposure and 3 – 7 days of growth under test conditions.
The concentrations of Oleyl dipropylene triamine were analysed at the concentration levels 0.32 and 3.20 mg/L (prepared without algae) and the control at test start via LC-MS/MS analysis. The measured concentrations at test start were in the range of 49 – 78 % of the nominal values. At the end of the test Oleyl dipropylene triamine was analysed at concentration levels 0.32 and 3.20 mg/L (prepared without algae) and 3.2 mg/L (prepared with algae). The measured concentrations were in the range of LOQ to 6% of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No adsorption ( LOQ) of the test item to glassware was observed. Therefore all effect values are given based on nominal concentrations of Oleyl dipropylene triamine (CAS No. 28872-01-7).
Executive summary:

The toxicity of Oleyl dipropylene triamine (CAS No. 28872-01-7) (Batch no.: S001256)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from May 26 to July 03, 2009with the definitive exposure phase being conducted between June 23 to 26, 2009. The aim of the study was the determination of EC10 - , EC20- and EC50 - values of growth rate and yield over a period of 72 h.

Oleyl dipropylene triamine(CAS no. 28872-01-7) is practically insoluble in water and also has a strong tendency to adsorb to negatively charged surfaces such as suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many cationic substances in general but long chain alkyl polyamines in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavaibility and exposure assessment for cationic surface-active substances like the polyamines as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavailability that occurs in the environment with cationic surface-active substances.

The aquatic ecotoxicity tests with polyamines were therefore performed in river water to allow a PECaquatic,bulk/PNECaquatic,bulkapproach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing 'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PECaquatic,bulkis compared with the PNECaquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

         

In order to class standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

         

For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier and more realistic, and if compared to PECbulk clearly provide a more appropriate assessment of risks for the environment.

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 approximately 5 x  103cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of 10, nominal: 0.10 - 0.32 - 1.00 - 3.20 - 10.0 mg/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 Oleyl dipropylene triamine were analysed at the concentration levels 0.32 and 3.20 mg/L (prepared without algae) and the control at test start via LC-MS/MS analysis. The measured concentrations at test start were in the range of 49 – 78 % of the nominal values. At the end of the test Oleyl dipropylene triamine was analysed at concentration levels 0.32 and 3.20 mg/L (prepared without algae) and 3.2 mg/L (prepared with algae). The measured concentrations were in the range of LOQ to 6% of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No adsorption ( LOQ) of the test item to glassware was observed. Therefore all effect values are given based on nominal concentrations of the test item. For details of the analytical results please refer to part 9.

Oleyldipropylene triamine (CAS No. 28872-01-7) 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 specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.518 (0.348 – 0.594) and 0.118 ( 0.10 – 0.155) mg/L, respectively. The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 1.09 (1.02 – 1.14) and 0.356 (0.314 – 0.398) mg/L, respectively.

After 72 h algae were transferred from the nominal concentration of 1.00 – 10.0 mg/L and the control to fresh untreated medium and allowed to grow for further 3 – 7 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 10.0 mg/L (highest test concentration)

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