Dimethyldioctylammonium chloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Reliable study carried out in accordance with OECD Guideline 201 and GLP compliant. There were no reported deviations from the methodology.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Also in accordance with US EPA OPPTS Number 850.5400

Deviations:

no

Principles of method if other than guideline:

In addition to the standard guideline, the test duration was extended to 96 hours and after 96 hours a recovery phase was observed for 6 days.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test substance Bardac 2280 containing 81% of didecyldimethyl ammonium chloride (DDAC) as the active ingrediant was provided by Lonza Inc. In addition, radio-labelled 14C-DDAC was received from Wizard Laboratories Inc with a reported purity of 99.88%.

Analytical monitoring:

yes

Details on sampling:

The freshwater alga was exposed to a geometric series of six test concentrations and a negative control under static conditions for 96 hours. Three replicate test chambers were maintained in each treatment and control group. Two replicate test chambers were also maintained with the absence of algae to verify test concentrations. Samples were collected from each replicate at test initiation and approximately 24 hour intervals during the test to determine cell densities for the calculation of growth inhibition.

Vehicle:

no

Details on test solutions:

A stock solution was prepared using 1.0 mCi of radio-labelled 14C-DDAC with 100 ml of NANOpure water. When analysed (98% recovery) the mean concentration was 326,418 dpm. This was further diluted to create a secondary stock solution used to prepare the test solutions and corresponded to 3.5 ug of the radio-labelled DDAC for each litre of test solution. In addition, non-labelled DDAC was prepared to a concentration of 0.20 ug DDAC/ml. The test medium was then prepared with equal volumes of the two stock solutions to prepare the 4.7, 9.4, 19, 38, 75 and 150 ug/L nominal test solutions. Test solutions were mixed by inversion. The potential for DDAC to bind to glassware was minmised by pre-treatment of the glassware with DDAC concentrations that corresponded to each treatment group, e.g. the 150 ug/l vessels were pretreated with 150ug/l DDAC solutions for 10 minutes. Several non-GLP trials were performed prior to the test to undertand binding potential to glassware. It was found that DDAC remained available to algae when bound to the glassware, hence the corresponding test concentration was used for pre-treatment.

Test organisms (species):

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

Details on test organisms:

The freshwater alga Selenastrum capricornutum was the test organism with original cultures obtained from the University of Toronto and further cultured in the laboratory's own facility. Algal cells were obtained from a culture that had been actively growing for at least two weeks prior to the test. The culture medium included a stock nutrient solution prepared by the laboratory with purified well water. The pH was adjusted to 7.5 +/- 0.1 using 10% HCl, where appropriate and sterlised by filtration before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

An exposure duration of 96 hours was used.

Post exposure observation period:

Following the 96 hour exposure duration, a recovery phase was observed for six further days by dilution of the test substance to a concentration that would not cause inhibition. Cell densities were counted at the initiation of the recovery phase, day 3 and day 6.

Hardness:

No information reported

Test temperature:

Temperature ranged from 23.2 to 24.4 degrees C

pH:

pH ranged from 7.2 to 7.3

Dissolved oxygen:

No information reported

Salinity:

No information reported

Conductivity:

No information reported

Nominal and measured concentrations:

Nominal concentrations were 0, 4.7, 9.4, 19, 38, 75 and 150 ug/L The mean measured DDAC concentrations were 0, 3.3, 6.86, 14.3, 29.9, 62.6 and 129.5 ug/L. The mean percentage of the nominal concentrations ranged from 70 to 86%.

Details on test conditions:

Test chambers were sterilised 250 ml Erlenmeyer flasks pretreated with DDAC (for each respective treatment concentration) and plugged with foam stoppers. Each contained 100 ml of test solution or control medium. Each vessel was labelled and randomly positioned daily on a mechanical shaker in an environmental chamber designed to maintain the test temperature. The flasks were shaken continuously at 100 rpm. The algae were under 24 hours fluorescent lighting. Environmental conditions were monitored twice daily.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

26 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

13 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Exponential growth was achieved and passed the OECD Guideline 201 QC criterion of a 16 fold increase in growth over 72 hours. After 96 hours of exposure to DDAC, inhibition of cell density in the 3.3, 6.9, 14, 30, 63 and 130 ug/L treatment groups was -0.93, 6.6, 7.8, 58, 98 and 99% respectively relative to the negative control. The 24-hour NOEC for cell density was 3.3ug DDAC/L. The 48, 72 and 96-hour NOEC was 14 ug DDAC/L. The 24-hour NOEC for biomass was 3.3 ug DDAC/L. The 48, 72 and 96-hour NOEC for biomass was 14 ug DDAC/L. After 96 hours of exposure there was no difference in cell shape, size or colour between any of the treatment groups and control replicates. In addtion, there was no evidence of aggregation or flocculation of cells, nor adherence to the test chambers.

In the study, 63 and 130 ug/L treatment groups were maximally inhibited at the end of the 96 hours exposure. By day 6 of the recovery phase, sufficient growth was observed in all relicates to indicate recovery from exposure to the test substance.

Results with reference substance (positive control):

No information reported

Reported statistics and error estimates:

Statistical analyses were conducted using the SAS system for Windows version 8.0. Cell densities and the area under the growth curve were statistically analysed by non-linear regression versus concentration to determine EC10 and EC50 values, including 95% confidence limits. Cell density and area under the growth curve were evaluated for normality and homogeneity of variance 9p=0.05) using the Shapiro-Wilk's and Levene's tests, respectively, and were compared to the negative control using analysis of variance (ANOVA) and Dunnett's test (p=0.05).

A summary of the results is presented in the tables below:

72-hour ECx values expressed as DDAC equivalents (µg/l)	Cell density	Growth inhibition
EC10 (95% confidence limits)	11 (8.2 to 16)	11 (8.0 to 16)
EC50 (95% confidence limits)	27 (22 to 32)	27 (22 to 32)
96-hour ECx values expressed as DDAC equivalents (µg/l)
EC10 (95% confidence limits)	14 (9.0 to 21)	13 (9.2 to 18)
EC50 (95% confidence limits)	26 (21 to 33)	26 (22 to 32)

NOEC expressed as DDAC equivalents (µg/l)	Cell density	Growth inhibition
24 hour	3.3	3.3
48 hour	14	14
72 hour	14	14
96 hour	14	14

Validity criteria fulfilled:

yes

Conclusions:

In a 96-hour static toxicity study of Selenastrum capricornutum exposed to the test substance (DDAC or didecyldimethyl ammonium chloride) resulted in a 72-hour and 96-hour EC50 of 27 and 26 ug/L as a measured concentration, respectively, for the inhibition of algal growth. The corresponding NOEC was 14 ug/L (both durations) for growth inhibition.

Executive summary:

A toxicity study of the test substance DDAC exposed to the freshwater algae Selenastrum capricornutum in a static test resulted in a 72-hour EC50 of 27 ug/L (0.027 mg/L) based on a growth inhibition endpoint. A NOEC of 14 ug/L (growth inhibition) was observed. The study was extended to observe recovery following exposure to the test substance and based on growth the algae were seen to recover. The reliable study corresponds to the OECD Test Guideline 201 and was undertaken by a commercial laboratory using its own algal culture. No adverse effects was observed in the control groups of organisms.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16/10/2017 to 19/10/2017 (experimental exposure)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Reliable study carried out in accordance with OECD Guideline 201 and GLP compliant. There were no reported deviations from the methodology.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch no.of test material: 6021-038
- Expiration date of the lot/batch: 23/05/2019
- Purity: 95.72%
- Manufacture date: 23/05/2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature, protected from light
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all concentrations were sampled at 0 and 72 hours. 72 h samples contained algae
- Sample storage conditions before analysis: All 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: Nominal 10 mg/L test substance was prepared in dilution water, and shaken until visually clear. 5 concentrations 0.0100, 0.0316, 0.100, 0.316 and 1.00 mg/L were prepared
- Eluate: OECD TG 201 medium
- Controls: Dilution water only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No data

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Preculture prepared in dilution water

ACCLIMATION
- Any deformed or abnormal cells observed: None observed at the start of the exposure period

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Light intensity: 4796-5887 (lux), mean 5531 lux

Post exposure observation period:

Not applicable

Hardness:

Nominally 0.24 mmol Ca+Mg/L

Test temperature:

21.5-22.5 oC; mean 22.0 oC

pH:

0h: 8.07-8.18
72h: 8.08-8.81

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Control and nominal 0.0100, 0.0316, 0.100, 0.316 and 1.00 mg/L
0h measured concentrations were in the range 103-114% of nominal
72h measured concentrations were in the range 105-117% of nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile 250 mL Erlenmeyer flasks
- Type (delete if not applicable): Closed with wool plugs
- Fill volume: 100 mL
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Initial cells density: 5 x 103 – 104 cells/mL (nominal), 6839 cells/mL (actual)
- Control end cells density: 1.5 x 106 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD TG 201 medium
- Culture medium different from test medium: Nutrient medium Z according to Luettge et al (1994)
- Intervals of water quality measurement: No data

OTHER TEST CONDITIONS
- Adjustment of pH: No data
- Photoperiod: 24h/day light
- Light intensity and quality: 4440-8880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Cell density measured daily by Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.0100, 0.100, 1.00 and 10.0 mg/L (nominal) as test substance
- Results used to determine the conditions for the definitive study:
Nominal conc (mg/L) Growth rate inhibition (%) Yield inhibition (%)
0.0100 0 2
0.100 22 73
1.00 100 100
10.0 100 100

Reference substance (positive control):

yes

Remarks:

Potassium chromate; determined over the period 10/10/2017 to 13/10/2017

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 0.122 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI = 0.109-0.187 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.01 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 0.032 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI = 0.0248-0.0396 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.01 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): None observed at 0 or 72h
- Any stimulation of growth found in any treatment: None observed

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 0.535 mg/L (growth rate); 0.320 mg/L (yield)
- Results were within validity range of test facility refrence tests

Endpoint	Growth rate inhibition (mg/L)	95% CI	Yield inhibition (mg/L)	95% CI
NOEC	0.0100	-	0.0100	-
EC10	0.0581	-	<0.0100	-
EC50	0.122	0.109-0.187	0.0318	0.0248-0.0396

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 (growth rate) value and the 95% confidence intervals were determined to be 0.122 (0.109-0.187) mg/L and the EyC50 (yield / biomass) value and the 95% confidence intervals were determined to be 0.0318 (0.0248-0.0396) mg/L, based on nominal test concentrations

Executive summary:

In a 72-hour toxicity study the green alga, Pseudokirchneriella subcapitata, was exposed to a range of test concentrations under static conditions in accordance with the OECD test guideline 201 and in compliance with GLP. The nominal test concentrations were a control and 0.0100, 0.0316, 0.100, 0.316 and 1.00 mg/L. Measured concentrations were within 20% of nominal, therefore, the results were calculated using nominal test concentrations.

Under the conditions of the test, the ErC50 (growth rate) was determined to be 0.122 mg/L and the EyC50 (yield / biomass) was determined to be 0.0318 mg/L, based on nominal test concentrations. The NOEC was determined to be 0.010 mg/L for growth inhibition and yield (biomass).

This toxicity study is considered acceptable and satisfies the guideline requirements for an algal growth inhibition test.

Description of key information

A reliable study (Klimisch 1) is available for test substance dioctyldimethylammonium chloride, C8DAQ (Klix, 2017). The test substance was exposed to the freshwater alga, Pseudokirchneriella subcapitata, in a 72-hour static test. An ErC50 (growth rate inhibition) of 0.122 mg/L was determined based on nominal test concentrations. A NOEC of 0.01 mg/L (growth rate inhibition) was observed. The study was conducted according to the OECD test guideline 201, in compliance with GLP. The study was conducted by a commercial laboratory using its own algal culture.

A reliable study (Klimisch 1) for the read-across substance DDAC using the freshwater alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) is available (Desjardins, 2002). This study determined a 72-hour EC50 of 0.027 mg/L (27 ug/L) and a NOEC of 0.014 mg/L (14 ug/L) based on growth inhibition. The study was extended and growth was observed to recover. The study corresponds to the OECD Test Guideline 201 and was undertaken by a commercial laboratory using its own algal culture.

Two reliable studies (Klimisch 1) are available on the read-across substance N,N-Didecyl-N,N-dimethylammonium carbonate (DDACarbonate) (Safepharm, 2004; Wildlife International, 2004). In the Safepharm study (2004), the diatom, Skeletonema costatum, after 96 hours of exposure, resulted in a cell density percent inhibition in the 14, 20, 26, 40 and 62 μg AI/L treatment groups was 0.16, 1.3, 64, 56 and 97 %, respectively, relative to the negative control. The 96-hour EC50 for cell density was determined to be 25 µg/L (24 – 26 µg/L), the 96-hour NOAEC was 20 μg AI/L. Aliquots of the 62 µg AI/L test solution were diluted with algal medium and cultured for up to 9 days. However, based on the growth observed over the recovery phase, the effect on algal growth was found to be more algistatic than algicidal.

In the Wildlife International study (2004), OECD 201 test, Scenedesmus subspicatus was exposed to an aqueous solution of the read across substance DDACarbonate at mean measured concentrations of 0, 0.0191, 0.0502, 0.152, 0.353 and 0.798 mg AI/L. Stability analyses identified a decline in measured test concentrations throughout the test was likely due to adsorption to algal cells and was in line with the recovery analyses performed with the addition of algal cells. Overall the decline in measured concentrations followed a concentration dependent pattern with greater losses being observed at the lower test concentrations. Given that there was a decline in measured test concentrations over the test period, it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. In terms of the pure active ingredient (as 100 % AI) the 72-hour EbC50 was determined 0.022 mg AI/L (0.020 – 0.026 mg AI/L), the 72-hour ErC50 was determined 0.035 mg AI/L (0.029 – 0.042 mg AI/L) and the No Observed Effect Concentration (NOEC) at 72 hours 0.0152 mg AI/L.

The algal study data for C8DAQ, DDAC and DDACarbonate (2 studies) determined ErC50 (growth rate inhibition) of 0.122 mg/L, 0.027 mg/L, 0.025 mg/L, 0.035 mg/L, respectively and NOEC’s of 0.010 mg/L, 0.014 mg/L, 0.020mg/L and 0.0152 mg/L, respectively. It is recommended that the algal study for C8DAQ (Klix, 2017) is the key study endpoint considered in PNEC development based on its reliability and the use of the test substance C8DAQ in toxicity testing.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.122 mg/L

EC10 or NOEC for freshwater algae:

0.01 mg/L