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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-09-03 to 2012-09-12
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
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0100 - 0.0250 - 0.0625 - 0.156 - 0.391 mg/L
- Sampling method: Each test item concentration and the control were analytically verified at the beginning and the end of the test via LC-MS/MS analysis. At test start and end samples were taken out of additional replicates provided without algae. Sorption of the test item to the glassware of the test vessel was determined in one replicate of a concentration level close to the ErC10.
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (1.00 g/L) was prepared with demineralised water. For preparation of the stock solution the pH-value of the demineralised water was adjusted to pH 2 ± 0.1 by adding 1M HCl and NaOH. Dilutions of the stock solution were made with demineralised water without pH adjusting.
The stock solution was stirred at approx. 500 rpm for 24 hours at
50 °C. The stock solution was cooled down to ambient temperature for preparation of the test concentrations. The stock solution was a homogenous and clear solution.
- Eluate: demineralised water
- Differential loading: 0.0100 - 0.0250 - 0.0625 - 0.156 - 0.391 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- 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
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
After 72 hours algae were transferred from the three highest dosage level (nominal test item concentrations of 0.0625 to 0.391 mg/L) and the control into fresh untreated medium. 1.0 to 5.0 mL algae suspension from each test replicate and from two control replicates were filled up with dilution water to 100 mL and allowed to grow for further 3 to 5 days to determine whether the effect of the test item was reversible, respectively.
Hardness:
Not measured
Test temperature:
Mean: 22.3 °C, Min: 22.0 °C, Max: 22.5 °C
pH:
Nominal concentration Geometric mean measured concentration pH-value
[mg test item/L] [mg test item/L] Start; 0 hours End; 72 hours
0.391 0.427 7.98 8.26
0.156 0.153 7.98 8.29
0.0625 0.0583 7.97 8.45
0.0250 0.0220 7.96 8.67
0.0100 0.00759 7.92 8.68
Control 7.93 8.68
Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to information on materials and methods
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with 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: 5407 cells/mL
- Control end cells density: Mean 1743891 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:
According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1  0.2
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.

- Test medium: Water of the river Innerste was used. This river is located near D-31157 Ruthe-Sarstedt, Germany (Latitude 52.25°, Longitude 9.83°). Water parameters are given. Additionally 50 % of the components concentrations of the dilution water (total application volume 6.5 mL/L) acc. to the guideline was added to enable a sufficient growth of algae.
Water Parameters of the River Water

River Innerste
Location Amtshof K514, km 26,
31157 Ruthe-Sarstedt, Germany
Latitude:52.25°, Longitude: 9.83°
Sampling Date 2012-03-20
Weather on Day of Sampling Sunny, slightly clouded; approx. 15°C
Colour Yellowish to brownish, clear
pH 7.93
Conductivity [µS/cm] 856
Dissolved Oxygen [mg O2/L] 11.2
DOC [mg C/L] 2.80*
TOC [mg C/L] 3.66*
Ammonium-N [mg N/L] 0.07
Nitrate-N [mg N/L] 3.40
Total Nitrogen [mg N/L] 3.80
o-Phosphate-p [mg P/L] < 0.20
Total Phosphate [mg P/L] < 0.20
Suspended Matter [mg/L] 14.0
Total Hardness [mg CO3/L] 271
Storage conditions 15 ± 2°C


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 hours)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition[%] Yield Inhibition [%]
10.0 100 100
1.00 100 100
0.1 44 89
0.01 1 4
0.001 0 -2













Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
44.2 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (38.5 - 49.7)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
42.1 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
95.3%
Basis for effect:
growth rate
Remarks on result:
other: (36.7 - 47.4)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
86.8 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (81.0 - 93.9)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
82.7 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
95.3%
Basis for effect:
growth rate
Remarks on result:
other: (77.2 - 89.5)
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: 0.718 (CI 0.699 - 0.737) mg/L
Yield Inhibition: 0.422 (CI 0.386 - 0.466) mg/L
Reported statistics and error estimates:
NOEC/LOEC were determined by calculation of statistical
significance of growth rate and yield. As 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. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. 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. For growth rate the two highest concentration levels (both with > 80 % inhibition) and for yield data the highest concentration level (100 % inhibition) have been excluded to achieve normally distributed data.
Detection of statistically not significant differences were considered positive when inhibition values were ≥ 10 %. These findings were marked “biologically significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.

Cell Densities

Nominal concen-tration

Geometric mean measured concen-tration

Replicate

Cell density [cells/mL]

[mg test item/L]

[mg test item/L]

No.

0 hours

24 hours

48 hours

72 hours

0.391

0.427

1

5407

5127

2314

4560*

2

5407

5273

LOQ

6840*

3

5407

6746

2463

4940*

Mean

5407

5715

n.a.

5447

0.156

0.153

1

5407

15938

12639

7096

2

5407

16617

19205

17116

3

5407

14487

12909

11483

Mean

5407

15681

14918

11898

0.0625

0.0583

1

5407

28835

89792

373592

2

5407

23933

106048

457412

3

5407

30562

91430

391340

Mean

5407

27777

95757

407448

0.0250

0.0220

1

5407

37468

276704

1351556

2

5407

35623

359051

1512655

3

5407

35699

282686

1673882

Mean

5407

36263

306147

1512698

0.0100

0.00759

1

5407

37992

327250

1659340

2

5407

39462

330844

1620605

3

5407

43624

370024

1599015

Mean

5407

40359

342706

1626320

Control

1

5407

42548

319122

1553105

2

5407

44354

392414

1609683

3

5407

53422

429345

1909340

4

5407

43529

417179

1609937

5

5407

53314

357171

2002177

6

5407

42094

373910

1779101

Mean

5407

46544

381524

1743891

Evaluation after 72 hours

              Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal concen-tration

Geometric mean measured concen-tration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg test item/L]

[mg test item/L]

No.

[d-1]

[%]

[cells/mL]

[%]

0.391

0.427

1

-0.057

100

-847

100

2

 0.078

  95.9

1433

  99.9

3

-0.030

100

-467

100

Mean

*

-0.003

  98.6

*

40

100

0.156

0.153

1

 0.091

  95.3

1689

  99.9

2

 0.384

  80.0

11709

  99.3

3

 0.251

  87.0

6076

  99.7

Mean

*

 0.242

  87.4

(+)

6491

  99.6

0.0625

0.0583

1

 1.41

  26.6

368185

  78.8

2

 1.48

  23.1

452005

  74.0

3

 1.43

  25.8

385933

  77.8

Mean

(+)

 1.44

  25.2

(+)

402041

  76.9

0.0250

0.0220

1

 1.84

    4.34

1346149

  22.6

2

 1.88

    2.39

1507248

  13.3

3

 1.91

    0.63

1668475

  4.03

Mean

(-)

 1.88

    2.45

(-)1

1507291

  13.3

0.0100

0.00759

1

 1.91

    0.78

1653933

    4.86

2

 1.90

    1.19

1615198

    7.09

3

 1.90

    1.42

1593608

    8.33

Mean

(-)

 1.90

    1.13

(-)

1620913

    6.76

Control

1

 1.89

1547698

2

 1.90

1604276

3

 1.96

1903933

4

 1.90

1604530

5

 1.97

1996770

6

 1.93

1773694

Mean

 1.92

1738484

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

2.06

2.02

1.58

1.89

0.265

14.0

19.1

2

2.11

2.18

1.41

1.90

0.424

22.3

3

2.29

2.08

1.49

1.96

0.414

21.2

4

2.09

2.26

1.35

1.90

0.483

25.4

5

2.29

1.90

1.72

1.97

0.289

14.6

6

2.05

2.18

1.56

1.93

0.329

17.0

Mean

1.92

 

SD ±

0.03

 

CV [%]

1.80

 

Inhibition of Growth Rate

Nominal                                [mg test item/L]

Geometric mean measured               [mg test item/L]

NOEC

0.0250

0.0220

LOEC

0.0625

0.0583

ErC10

0.0442 (0.0385 – 0.0497)

0.0405 (0.0350 – 0.0458)

ErC20

0.0571 (0.0525 – 0.0618)

0.0530 (0.0485 – 0.0576)

ErC50

0.0868 (0.0810 – 0.0939)

0.0824 (0.0766 – 0.0895)

Inhibition of Yield

NOEC

0.0100

0.00759

LOEC

0.0250

0.0220

EyC10

0.0209 ( 0.0100 – 0.0285)

0.0182 ( 0.00759 – 0.0253)

EyC20

0.0304 (0.0259 – 0.0365)

0.0271 (0.0229 – 0.0329)

EyC50

0.0458 (0.0405 – 0.0499)

0.0419 (0.0368 – 0.0459)

Validity criteria fulfilled:
yes
Conclusions:
In this study 3-(Isodecyloxy)propylamine was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values: The nominal EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0868 (0.0810 – 0.0939) mg test item/L and 0.0458 (0.0405 – 0.0499) mg test item/L, respectively. The nominal EC10-values after 72 hours for inhibition of growth rate and inhibition of yield were 0.0442 mg test item/L and 0.0209 mg test item/L, respectively. The geometric mean measured EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0824 (0.0766 – 0.0895) mg test item/L and 0.0419 (0.0368 – 0.0459) mg test item/L, respectively. The geometric mean measured EC10-values after 72 hours for inhibition of growth rate and inhibition of yield were 0.0405 mg test item/L and 0.0182 mg test item/L, respectively. For other endpoints, please refer to Table 1. All effect values are given based on the nominal and geometric mean measured concentrations of 3-(Isodecyloxy)propylamine.
Executive summary:

The toxicity of 3-(Isodecyloxy)propylamine (CAS no. 30113-45-2, batch no.: 890000022898) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The test period was between 2012-09-03 and with the definitive exposure phase from to at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany . The aim of the study was the determination of NOEC, LOEC, EC10 -, EC20- and EC50 - values of growth rate and yield in natural river water over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5407 cells/mL. Five concentration levels were tested in a geometrical series with a dilution factor of 2.5 (nominal): 0.0100 - 0.0250 - 0.0625 - 0.156 - 0.391 mg/L, corresponding to geometric mean measured concentrations of 0.00759 - 0.0220 - 0.0583 - 0.153 - 0.427 mg/L. Three replicates were tested for each concentration level and six replicates for the control. Environmental conditions (pH and room temperature) were determined to be within the acceptable limits.

Various concentration levels of the test item and the control were analytically verified via LC-MS/MS from freshly prepared media after 0 and from aged media after 72 hours. The recovery rates of 3-(Isodecyloxy)propylamine in freshly prepared media were in the range of 99 – 116 % of the nominal values. In old test media the test item gave recoveries of 54 - 103 % of the nominal values. The adsorption to the test vessels after 72 hours of exposure was determined of the dosage level close to the ErC10 with a geometric mean measured concentration of 0.0583 mg/L of the test item. The absorbed amount of the test item was 0 % of the applied amount. For details of the analytical results please refer to part 4.2.3 and 9. All effect values are given based on the nominal and geometric mean measured concentrations of 3-(Isodecyloxy)propylamine.

NOEC, LOEC, EC-Values and 95 % Confidence Intervals of
3-(Isodecyloxy)propylamine  (0-72 hours)

                   based on nominal and geometric mean measured test item concentrations [mg/L]

Inhibition of Growth Rate

Nominal                                [mg test item/L]

Geometric mean measured               [mg test item/L]

NOEC

0.0250

0.0220

LOEC

0.0625

0.0583

ErC10

0.0442 (0.0385 – 0.0497)

0.0405 (0.0350 – 0.0458)

ErC20

0.0571 (0.0525 – 0.0618)

0.0530 (0.0485 – 0.0576)

ErC50

0.0868 (0.0810 – 0.0939)

0.0824 (0.0766 – 0.0895)

Inhibition of Yield

NOEC

0.0100

0.00759

LOEC

0.0250

0.0220

EyC10

0.0209 ( 0.0100 – 0.0285)

0.0182 ( 0.00759 – 0.0253)

EyC20

0.0304 (0.0259 – 0.0365)

0.0271 (0.0229 – 0.0329)

EyC50

0.0458 (0.0405 – 0.0499)

0.0419 (0.0368 – 0.0459)

After 72 hours algae were transferred from the three highest dosage levels (nominal: 0.0625 – 0.391 mg/L and geometric mean measured: 0.0583 – 0.427 mg/L) and from the control to fresh untreated medium and allowed to grow for further 3 to 5 days under test conditions. Cell growth occurs at these concentrations. Therefore, there is potential for recovery following exposure up to the nominal test item concentration of 0.391 mg/L corresponding to the geometric mean measured test item concentration of 0.427 mg/L (highest test concentration).

Description of key information

The ErC50 of 0.0827 mg/L and the ErC10 of 0.0421 mg/L were determined in a test according to OECD TG 201 (GLP, Reliability 1)  with 3-(Isocedyloxy)propylamine using river water. Effect concentrations are based on active ingredient.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.083 mg/L
EC10 or NOEC for freshwater algae:
0.042 mg/L

Additional information

Two algae toxicity studies are available for 3-(Isodecyloxy) propylamine and 3-(Isodecyl)propylamine acetate respectively. The substances have the same chemical structure with the only difference being the presence of acetate. Acetate is not expected to have an influence on the aquatic toxicity. Therefore the results have been read-across. As the effect concentrations are within the same order of magnitude for the two studies (actually less than a factor of 2 -3 difference), the lowest effect concentrations (3-(Isodecyloxy) propylamine have been selected as key value for toxicity towards algae.

The studies have been performed according to OECD TG 201 under GLP. The studies were assigned reliability 1, because all validity criteria were fulfilled. The studies were performed with river water, rendering the test results suitable for the use of the bulk water approach that will be used for the chemical safety assessment. The 72h ErC50 was 0.0827 mg/L a.i for the 3 -(Isodecyl)propyl amine and 0.15 mg/L for the 3-(Isodecyl)propylamine acetate respectively. The 72h ErC10 were 0.0421 mg/L a.i. and 0.0852 mg/L a.i. respectively.

In the key study the toxicity to Pseudokirchneriella subcapitata was determined according to OECD TG 201 (Noack, 2012). The concentrations were analytically verified and the measured concentrations at test start were in the range of 99 – 116% of the nominal values. At the end of the test the levels ranged from 54 – 103% of the nominal values. Biodegradation is not expected to occur to a large extent due to the short timeframe of the test. The observed decrease is attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. Negligible adsorption (1% of nominal concentration) of the test item to glassware was measured. All effect values are given based on nominal concentrations as well as measured concentrations of the test item. The nominal effect concentrations have been selected as key value for the PNEC derivation in order to render the PNEC suitable for use in the bulk approach.

After 72 h, algae were transferred from the three highest dose levels (nominal concentrations 58 – 427 µg/L) and from the control to fresh untreated medium and allowed to grow for a further 3-5 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 427 µg/L (highest test concentration).