3,3'-ethylenedioxybis(propylamine)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-27 - 2017-10-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD guideline 201 and under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016/266

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD No. 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP Monitoring Authorities, Date of decission 2015 - 11- 16

Analytical monitoring:

yes

Details on sampling:

Sampled concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: duplicate samples were taken from the test media of all test concentrations
and the control at the start of the test (without algae) and at the end of the test (containing algae).
For sampling at the end of the test, the test medium of the treatment replicates was pooled.
- Sample storage conditions before analysis: all samples were frozen (at -20 ± 5 °C)
- Analysed samples: The concentrations of the test item JEFFAMINE® EDR-176 were determined in one
of the duplicate samples from all test concentrations from both sampling times (0 and 72 hours). From
the control samples, one of the duplicate samples was analyzed per sampling time.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by
dissolving 101 µL of the test item completely in 1000 mL of test water. This volume is equivalent to
a loading rate of 100 mg/L, considering the relative density of the test item of 0.99. The test item
was dissolved by intense stirring for 15 minutes at room temperature. During this stirring period
the pH was adjusted from pH 10 to 7.5 with 1 M hydrochloric acid solution.
- Eluate: The highest test concentration of 100 mg/L was further diluted with test water to prepare
the test media with the lower test concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): After the dissolving procedure
the highest test concentration of 100 mg/L was a clear solution.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (Raphidocelis subcapitata;
formerly Selenastrum capricornutum)
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute
for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up three days before
the start of the exposure.
- Method of cultivation: The algae were cultivated at IES Laboratories under standardized
conditions according to the test guidelines. The algae were cultivated under the test conditions
and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Acclimation period: no acclimatization was necessary since the algae were cultivated under the
same conditions as in the test.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

other: AAP-medium according to the guideline

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

not performed

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

24°C

pH:

7.5-8.1

Dissolved oxygen:

n.d.

Salinity:

n.d.

Conductivity:

n.d.

Nominal and measured concentrations:

nominal concentrations / 0 hours / 72 hours / mean measured concentration (geometric mean)
[mg/L]
0.32 / 0.193 / 0.136 / 0.16
1.0 / 0.772 / 0.691 / 0.73
3.2 / 2.66 / 2.88 / 2.8
10 / 9.89 / 9.33 / 9.6
32 / 34 / 35 / 34
100 / 101 / 102 / 101

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glas, 75 mL, open, 30 mL
- Aeration: No
- Initial cells density: 5000 cells/mL (corresponding 0.83E+04 relative fluorescence)
- Control end relative fluorescence: 222.8E+04
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP-Medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Macro-nutrients: Ingredients / Concentration
NaHCO3 / 15.0 mg/L
K2HPO4 / 1.044 mg/L
MgSO4 × 7 H2O / 14.6 mg/L
MgCl2 × 6 H2O / 12.16 mg/L
CaCl2 × 2 H2O / 4.41 mg/L
NaNO3 / 25.5 mg/L
Trace elements: Ingredients / Concentration
H3BO3 / 186.0 µg/L
MnCl2 × 4 H2O / 415.0 µg/L
ZnCl2 / 3.27 µg/L
CoCl2 × 6 H2O / 1.43 µg/L
CuCl2 × 2 H2O / 0.012 µg/L
Na2MoO4 × 2 H2O / 7.26 µg/L
FeCl3 × 6 H2O / 160.0 µg/L
Na2EDTA × 2 H2O: 300.0 µg/L

- CaCO3: 15 mg/L
- Water hardness: 0.15 mmol/L (= 15 mg/L as CaCO3)
- Culture medium different from test medium: No
- Intervals of water quality measurement: it is a reconstituted test water

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: The stock solution 100 mg/L the pH was adjusted from pH 10 to pH 7.5 with 1M HCl
- Photoperiod: continuously illuminated
- Light intensity and quality: 70-74 µEs-1m-2, LED

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of biomass: fluorimeter (0, 24, 48 and 72 hours). (For the inoculum culture: electronic
particle counter for determination of cell concentration)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Justification for using less concentrations than requested by guideline: the range-finding test showed a
flat concentration-effect relationship

RANGE-FINDING STUDY without pH adjustment:
- Test concentrations: 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study:
Concentration / Inhibition of Average Growth after 72 hours / Inhibition of Yield after 72 hours / pH test start / pH test end
1.0 mg/L / 1.4 % / 7.3% / 7.3 / 7.5
10 mg/L / 7.6 % / 34 % / 7.6 / 7.8
100 mg/L / 62 % / 97 % / 10 / 8.1

RANGE-FINDING STUDY with pH adjustment: The pH was adjusted in the 100 mg/L stock solution from pH10 to pH 7.5
- Test concentrations: 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study:
Concentration / Inhibition of Avarage Growt after 72 hours / Inhibition of Yield after 72 hours / pH test start / pH test end
1.0 mg/L / 1.8 % / 9.5% / 7.4 / 7.9
10 mg/L / 4.9 % / 24 % / 7.4 / 8.0
100 mg/L / 23 % / 71 % / 7.4 / 7.9

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, tested as reference study under GLP twice a year.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 101 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: > the highest tested concentration

Remarks:

95 % Confidence Interval could not be determined

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % Confidence Interval : 28 – 37 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % Confidence Interval 31 – 41 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % Confidence Interval 3.0 – 5.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Inhibition of growth rate was 30 % at the highest tested concentration, i.e. mean measured concentration of 101 mg/L.
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no difference between the algae growing at the highest
concentration of 100 mg/L and the algae cells in the control
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72-hour EC50 for growth rate: 1.0 mg potassium dichromate/L.

Reported statistics and error estimates:

Growth rate and yield were calculated for each test flask. The mean values for growth rate and
yield were calculated for each treatment.

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and
their 95 % confidence intervals were calculated by Probit Analysis using linear maximum likelihood
regression.

For the determination of the LOEC and NOEC, the average growth rate and yield at the test
concentrations were compared to the control values by Williams t-test and Welch t-test with
Bonferroni-Holm-adjustment, where appropriate.

Table: Analytical results

 

Nominal Test Item Concentration	Measured Concentration of the Test Item [mg/L]	Measured Concentration of the Test Item [mg/L]	Mean Measured Concentration (Geometric Mean)
[mg/L]	0 hours	72 hours	[mg/L]
0.32	0.193	0.136^(a)	0.16
1.0	0.772	0.691	0.73
3.2	2.66	2.88	2.8
10	9.89	9.33	9.6
32	34.0	35.0	34
100	101	102	101

LOQ:     Limit of quantification (LOQ = 0.16 mg test item/L).

(a):        concentration below lowest calibration level; estimated value by extrapolation of calibration curve

 

Table: Biological results

Nominal Test Item Concentration Mean Measured Test Item Concentration Repl. No. Biomass of Algae* Biomass of Algae* Biomass of Algae* [mg/L] [mg/L] 24 Hours 48 Hours 72 Hours Control Control 1 7.0 44.7 237.2     2 5.9 41.9 223.4     3 5.0 44.0 214.4     4 5.4 43.2 223.5     5 6.0 42.5 228.0     6 5.1 43.9 210.7     Mean 5.7 43.4 222.8     SD 0.7 1.0 9.5 0.32 0.16 1 7.5 44.7 226.2     2 6.1 43.1 238.0     3 6.4 37.2 214.2     Mean 6.7 41.6 226.1     SD 0.7 3.9 11.9 1.0 0.73 1 6.9 41.7 216.3     2 5.8 38.9 201.6     3 6.2 37.9 180.4     Mean 6.3 39.5 199.4     SD 0.6 1.9 18.0 3.2 2.8 1 6.1 36.1 177.0     2 6.3 35.8 186.5     3 5.6 31.7 178.1     Mean 6.0 34.5 180.5     SD 0.3 2.5 5.2 10 9.6 1 6.6 36.3 189.1     2 5.4 33.6 184.0     3 5.7 33.7 184.6     Mean 5.9 34.5 185.9     SD 0.6 1.6 2.8 32 34 1 4.4 24.7 121.3     2 4.2 22.2 127.0     3 3.8 23.2 148.8     Mean 4.1 23.3 132.4     SD 0.3 1.3 14.5 100 101 1 2.4 6.0 35.4     2 1.7 7.1 41.2     3 1.9 6.5 45.7     Mean 2.0 6.5 40.8     SD 0.35 0.55 5.16

 SD:  Standard deviation

*:     The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 10000). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.83 x 10000 relative fluorescence units. 

Table: Average Growth Rates(µ)   Nominal Test Item Conc. Mean Measured Conc. Average Growth Rate µ (day^-1) Inhibition of µ (Ir) Average Growth Rate µ (day^-1) Inhibition of µ (Ir) Average Growth Rate µ (day^-1) Inhibition of µ (Ir) 0-24 h 0-24 h 0-48 h 0-48 h 0-72 h 0-72 h [mg/L] [mg/L] µ Ir[%] µ Ir[%] µ Ir[%] Control --- 1.929 0.0 1.979 0.0 1.865 0.0 0.32 0.16 2.081 -7.9 1.957 1.1 1.870 -0.3 1.0 0.73 2.027 -5.1 1.932* 2.4 1.827* 2.0 3.2 2.8 1.982 -2.7 1.864* 5.8 1.795* 3.8 10 9.6 1.958 -1.5 1.865* 5.8 1.805* 3.2 32 34 1.606* 16.7 1.669* 15.7 1.690* 9.4 100 101 0.866* 55.1 1.032* 47.8 1.297* 30.4 *:           Mean value statistically significantly lower than in the control (according to a Williams t-test one-sided smaller,a= 0.05).

Validity criteria fulfilled:

yes

Remarks:

Biomass increased by a factor of 269 over 72h. Mean coeff. of variation of the daily growth rates (section-by-section) was 12% (72h). Coeff. of variation of the average specific growth rates in the replicates was 0.8% (72h)

Conclusions:

In a valid, reliable and conclusive study according to OECD 201 (2006/2011), the impact of the test substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata is summarized in the table below. The results are based on mean measured concentrations (geometric mean) of the test item:

Growth rate:
EC10 (0 -72 h) [mg/L]: 33 (95 % confidence interval 28 -37)
EC20 (0 -72 h) [mg/L]: 63 (95 % confidence interval 58 -67)
EC50 (0 -72 h) [mg/L]: >101 (95 % confidence interval not determined)
NOEC [mg/L]: 0.16
LOEC [mg/L]: 0.73

Yield:
EC10 (0 -72 h) [mg/L]: 4.2 (95 % confidence interval 3.0 -5.5)
EC20 (0 -72 h) [mg/L]: 8.7 (95 % confidence interval 6.9 -11)
EC50 (0 -72 h) [mg/L]: 36 (95 % confidence interval 31 -41)
NOEC [mg/L]: 0.16
LOEC [mg/L]: 0.73

Executive summary:

The impact of the test substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3.

The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

The measured concentrations of the test substance in the test media of the test concentrations of 0.32 to 100 mg/L were between 60 and 106 % of the nominal values at the start of the test and 42 and 110 % at the end of the test. For the four higher levels in the range of nominal 3.2 to 100 mg/L the recoveries were between 83 to 106 % in the freshly prepared samples, and between 90 to 110 % in the aged samples. This shows the correct preparation of these samples and the stability of the test item during the test. For the test samples with nominal 0.32 and 1.0 mg/L lower recoveries of the test item were obtained, which could be due to adsorption of the test item on the surfaces of the vessels used in the test. During the test period of 72 hours, the test item concentration in the test media of the lowest concentration of 0.32 mg/L decreased to 70 % of the initial values. Therefore, the biological results are based on the mean measured concentrations. These were calculated as the geometric mean of the concentrations measured at the start and the end of the test.

Nominal Test Item Concentration: 0.32, 1.0, 3.2, 10, 32, 100 mg/L

Mean Measured Concentrations: 0.16, 0.73, 2.8, 9.6, 34, 101 mg/L

Growth rate:

EC10 (0 -72 h) [mg/L]: 33 (95 % confidence interval 28 -37)

EC20 (0 -72 h) [mg/L]: 63 (95 % confidence interval 58 -67)

EC50 (0 -72 h) [mg/L]: >101 (95 % confidence interval not determined)

NOEC [mg/L]: 0.16

LOEC [mg/L]: 0.73

Yield:

EC10 (0 -72 h) [mg/L]: 4.2 (95 % confidence interval 3.0 -5.5)

EC20 (0 -72 h) [mg/L]: 8.7 (95 % confidence interval 6.9 -11)

EC50 (0 -72 h) [mg/L]: 36 (95 % confidence interval 31 -41)

NOEC [mg/L]: 0.16

LOEC [mg/L]: 0.73

Description of key information

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No. 2016/266, C3.

The biological results are based on the mean measured concentrations.

These were calculated as the geometric mean of the concentrations measured at the start and the end of the test.

Nominal Test Item Concentration: 0.32, 1.0, 3.2, 10, 32, 100 mg/L

Mean Measured Concentrations: 0.16, 0.73, 2.8, 9.6, 34, 101 mg/L

Growth rate:

EC10 (0 -72 h) [mg/L]: 33 (95 % confidence interval 28 -37)

EC20 (0 -72 h) [mg/L]: 63 (95 % confidence interval 58 -67)

EC50 (0 -72 h) [mg/L]: >101 (95 % confidence interval not determined)

NOEC [mg/L]: 0.16

LOEC [mg/L]: 0.73

Yield:

EC10 (0 -72 h) [mg/L]: 4.2 (95 % confidence interval 3.0 -5.5)

EC20 (0 -72 h) [mg/L]: 8.7 (95 % confidence interval 6.9 -11)

EC50 (0 -72 h) [mg/L]: 36 (95 % confidence interval 31 -41)

NOEC [mg/L]: 0.16

LOEC [mg/L]: 0.73

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

0.16 mg/L

Additional information

The test item is not toxic on algae.