N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine

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:

January 2018 - January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

yes

Remarks:

See remarks below

Principles of method if other than guideline:

Deviation No. 1 to the Study Plan (25 July 2018): Correction of a description mistake
Description of the modification The additional vessels for chemical analysis were prepared by using 6 mL test medium (instead of 6 mL algal inoculum suspension) for each treatment group.
Reason for the modification In the last line of the first column of the table in study plan section 4.1, the description “algal inoculum suspension” is wrong. As the samples for the TOC-analysis are without test organisms, in order to not disturb the TOC-analysis (section 5.1), 6 mL test medium was added in each additional vessel for chemical analysis of each treatment group.
Impact on the study The described modification does not impact the quality and reliability of the study negatively. By adding 6 mL test medium (instead of 6 mL algal inoculum suspension), the additional vessels for chemical analysis are prepared correctly without test organisms.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item name: PU-2018-788
Product name: POLYCAT 77 – PU-2018-788
Chemical name: N-[3-(Dimethylamino)propyl]-N,N',N'-Trimethylpropane-1,3-diamine
Molecular formula: C11H27N3
CAS number: 3855-32-1
Batch/Lot number: 1944471
Active substance: 100 % (theoretically)
Purity: 97 %
Carbon content: 65.6 % (calculated)
Stability: Stable in aqueous solution
Water solubility: Soluble (ca. 190 g/L at 25 °C)
Volatility: Vapour pressure 5.453 hPa at 21 °C
Appearance: Liquid at RT
Colour: Colourless
Storage conditions: Room temperature
Expiration date: 26 March 2019
Certificate of analysis: 05 July 2018
Origin: Evonik Nutrition & Care GmbH
Identification: Appearance: Liquid; Colour: Colourless; Batch: 1944471

Analytical monitoring:

yes

Details on sampling:

Analytically measured TOC concentrations in the test solutions after 0 h, 24 h, 48 h and 72 h exposure.
Two samples of 6 mL were taken from both additional vessels (without test organisms in order to not disturb the TOC-analysis) of treatment A, B, C, D, E and NC after 0 h, 24 h, 48 h and 72 h exposure: 4 samples per treatment and time.
Of the 4 samples per treatment and time, 2 were analysed directly after sampling using a TOCanalyser (Non Purgeable Organic Carbon [NPOC] method) and 2 are stored as retain samples in the freezer (≤ -18 °C) until finalization of the study.

Vehicle:

not specified

Details on test solutions:

The test was performed with OECD TG 201 medium according to OECD 201 (2006)

Test organisms (species):

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

Details on test organisms:

The test organism Raphidocelis subcapitata (Strain No. 61.81 SAG) originates from the Culture Collection of Algae at the University of Goettingen. The strain used for this study has been cultured in suspension culture at Hydrotox GmbH since July 2017. Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium (composition: 496 mg/L NaNO3, 39 mg/L K2HPO4, 75 mg/L MgSO4×7H2O, 36 mg/L CaCl2×2H2O, 58 mg/L Na2CO3, 10 mg/L Na2EDTA×2H2O, 3 mg/L citric acid, 3 mg/L iron citrate, 0.1144 mg/L H3BO3, 0.0724 mg/L MnCl2× 4H2O, 0.0088 mg/L ZnSO4×7H2O, 0.0032 mg/L CuSO4×5H2O, 0.0010 mg/L Na2MoO4×2H2O, 0.0016 mg/L CoCl2×6H2O) under axenic conditions to keep it in exponential growth.
Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Sigma, Steinheim, Germany, Lot no.: MKBF2111V, expiration date: January 2018). The recent quality testing was performed in December 2017 which resulted in an ErC50 (72 h) of 1.18 mg/L (CL 95 %: 0.97 - 1.42 mg/L; interlaboratory test result for ErC50: mean of 1.19 mg/L with SD of 0.27 mg/L) and an EyC50 (72 h) of 0.61 mg/L (CL 95 %: 0.42 – 0.91 mg/L).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None specififed

Hardness:

Not specified

Test temperature:

The temperature during the exposure was 21.9 – 23.2 °C (required: 21 - 24 °C, constant within ± 2 °C)

pH:

The pH was 7.9 – 8.0 in the control and 8.0 – 9.6 in the test item treatment

Dissolved oxygen:

Not specified

Salinity:

Freshwater

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations of 6.25 – 100 mg/L test item were used.
The measured TOC concentrations in the test item treatment were 100.0 – 108.7 % of the nominal TOC concentrations and stable during the exposure

A preliminary test without GLP was performed before start of this GLP-study. Nominal concentrations of 1, 10 and 100 mg/L test item were tested and resulted in inhibitions of -7.0 %, 37.2 % and 102.1 % for yield and -1.7 %, -8.2 % and 173.8 % for growth rate (72 h), respectively.

Details on test conditions:

Preparation of the test treatment and control:
The stock solution was prepared by adding 111.1 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 1000 mL test medium and shaking for 10 min using an overhead shaker.
This stock solution was used as highest test item concentration in the test. The further test item concentrations were prepared by diluting the stock solution with test medium.

Exposure conditions:
The test vessels were sealed with cellulose stoppers and kept in the light incubator (specified temperature: 21 – 24 °C, constant within ± 2 °C). They were placed on the shaking table with adhesive mats and continuously shaken with 100 rpm and an orbital diameter of 5 cm.
The test vessels were continuously illuminated by LEDs (warm white) with a light intensity of 88.5 µE m-² s-1 ± 8.0 %. The pH was measured at the start (0 h) and at the end (72 h) of the test.
The temperature was measured continuously.

Measurement of chlorophyll fluorescence:
The algal biomass was monitored by measuring the chlorophyll fluorescence after 0 h, 24 h, 48 h and 72 h. From each test vessel, 200 µL test solution was transferred into a 96-well microplate and the fluorescence measured with the fluorescence microplate reader at an excitation wavelength of
465 nm and an emission wavelength of 670 nm. Each measurement was conducted in duplicate. If the variation coefficient was > 10 %, the measurement was repeated. Test medium was measured as blank value and subtracted from the fluorescence values in the test vessels.

Inspection of cell morphology:
At the start and the end of the test, the appearance of the algae was examined microscopically.
Any noticeable deviations were documented.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34.99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Criteria of validity
- The biomass (cell concentration) in the control has increased by a factor of 29.3 and therefore ≥ 16 during the test period.
- The mean coefficient of variation for section-by-section growth rate (day 0 - 1, 1 - 2 and 2 - 3) in the control is 11.0 % and therefore ≤ 35 %.
- The coefficient of variation of average specific growth rate during the test period in the control replicates is 5.5 % and therefore ≤ 7 %.
The test is valid according to OECD Test Guideline 201 (23 March 2006).

Validity criteria fulfilled:

yes

Conclusions:

In an Algal Growth Inhibition Test with Raphidocelis subcapitata according to OECD 201, the 72hr EC50 for both growth rate and yield was determined as 34.99 mg/L. The NOEC for both growth rate and yield was determined as 25 mg/L.

Executive summary:

A four day old pre-culture was used as inoculum in the test. The algae were exposed to different concentrations of the test item in OECD TG 201 medium for 72 h.

A static test design was applied with an accompanying TOC analysis. The algal biomass was determined after 0 h, 24 h, 48 h and 72 h exposure by measuring the surrogate parameter chlorophyll fluorescence. In the present study, nominal concentrations of 6.25 – 100 mg/L test item

were used.

A preliminary test without GLP was performed before start of this GLP-study. Nominal concentrations  of 1, 10 and 100 mg/L test item were tested and resulted in inhibitions of -7.0 %, 37.2 % and 102.1 % for yield and -1.7 %, -8.2 % and 173.8 % for growth rate (72 h), respectively.

The test was performed with OECD TG 201 medium according to OECD 201 (2006).

Before the start of the test, 1.190 mL of the algae stock suspension was diluted with 48.810 mL test medium to obtain a concentration of 5 × 104 cells/mL. This pre-culture was incubated for 4 d at 21.8 – 23.7 °C and 88.5 µE m-² s-1 ± 8.0 % continuous lighting. For the start of

the test, the cell concentration was determined using the Coulter Counter, resulting in 101.356 × 105 cells/mL. To obtain a nominal inoculum concentration of 7 × 104 cells/mL, 1.380 mL pre-culture was diluted into 200 mL test medium.

The stock solution was prepared by adding 111.1 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 1000 mL test medium and shaking for 10 min using an overhead shaker.

This stock solution was used as highest test item concentration in the test.

Test vessels were sealed with cellulose stoppers and kept in the light incubator (specified temperature: 21 – 24 °C, constant within ± 2 °C). They were placed on the shaking table with adhesive mats and continuously shaken with 100 rpm and an orbital diameter of 5 cm.

The test vessels were continuously illuminated by LEDs (warm white) with a light intensity of 88.5 µE m-² s-1 ± 8.0 %. The pH was measured at the start (0 h) and at the end (72 h) of the test.

The temperature was measured continuously.

The algal biomass was monitored by measuring the chlorophyll fluorescence after 0 h, 24 h, 48 h and 72 h. From each test vessel, 200 µL test solution was transferred into a 96-well microplate and the fluorescence measured with the fluorescence microplate reader at an excitation wavelength of

465 nm and an emission wavelength of 670 nm. Each measurement was conducted in duplicate. If the variation coefficient was > 10 %, the measurement was repeated. Test medium was measured as blank value and subtracted from the fluorescence values in the test vessels.

At the start and the end of the test, the appearance of the algae was examined microscopically. Any noticeable deviations were documented.

As the measured test item concentrations are within ± 20 % of the nominal concentrations, according to OECD 201 (2006), all results are given in relation to the nominal test item concentrations.

The measured TOC concentrations in the test item treatment were 100.0 – 108.7 % of the nominal TOC concentrations and stable during the exposure.

In treatment group A, B and C, partially thickened algal cells were observed. The morphology of the algal cells in the control showed no obvious abnormality.

The effect concentrations after 72 h exposure were calculated with the statistical software ToxRat Professional 3.2.1 (Table 3; Appendix V). All results are given in relation to the nominal test item concentrations.

Description of key information

In an Algal Growth Inhibition Test with Raphidocelis subcapitata according to OECD 201, the 72hr EC50 for both growth rate and yield was determined as 34.99 mg/L. The NOEC for both growth rate and yield was determined as 25 mg/L. 

Key value for chemical safety assessment

EC50 for freshwater algae:

34.99 mg/L

EC10 or NOEC for freshwater algae:

25 mg/L