Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate

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:

25 September 2017 to 16 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity: >98%; Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Yellow Liquid

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Aqueous samples (8 mL) of freshly prepared test media (0 hour) and pooled expired test media (72 hours) were collected in duplicate.
- Sample storage conditions before analysis: Sample storage stability was demonstrated over a period of 7 days at -20˚C.
- Sample processing: Prior to analysis, the samples were diluted with acetonitrile, then diluted further with diluent (OECD medium:acetonitrile, 4:1, v/v) as required to bring the response within the calibration range.

Vehicle:

no

Details on test solutions:

Preliminary analysis in another study with this UVCB test substance indicated that the test substance was soluble in test media. Test solution preparation followed the method developed in that study. At the start of the test, the test substance was weighed onto a glass slide and the slide was added to OECD medium, which was stirred for ca 23 hours at a rate such that a slight dimple was formed at the surface. After the stirring period, the preparation was allowed to settle for ca 1 hour after which the aqueous phase was removed by careful mid-depth siphoning to give the 10 mg/L stock solution. Dilutions were prepared in EC media to give the 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L test concentrations. The control solution consisted only of OECD medium.

Test organisms (species):

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

Details on test organisms:

- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Oban, UK
- Age of inoculum (at test initiation): Concentrated, axenic liquid slope cultures stored under refrigeration (2-8 °C) for up to 8 months.
- Method of cultivation: Prior to testing, duplicate starter cultures were prepared and incubated under test conditions to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 x 10^4 cells/mL.

Test type:

static

Water media type:

other: OECD medium

Limit test:

no

Total exposure duration:

72 h

Hardness:

not specified

Test temperature:

20.6 to 22.8°C

pH:

7.87 to 8.90

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L
Measured concentrations: (0 hour)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile, autoclaved glass 250 mL Erlenmeyer (conical) flasks
- Fill volume: 100 mL
- Aeration: None; shaken continuously (100 rpm) to keep algae in suspension and facilitate transfer
of CO2.
- Renewal rate of test solution: Not applicable; static
- Initial cell density: 1 x 10^4 cells/mL
- Control end cell density: 82.9x 10^4 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; OECD medium

OTHER TEST CONDITIONS
- Intervals of water quality measurement: 0 and 72 hours; temperature also was recorded continuously using minimum/maximum thermometer.
- Sterile test conditions: Yes
- Adjustment of pH: None reported.
- Photoperiod: Constant light
- Light intensity and quality: 4890 – 6100 lux (0 hour); 4660 – 5100 lux (72 hours); under florescent lights, emitting white light across the visible portion of the spectrum (400 - 700 nm).

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: particle counter (Z2 Coulter Counter)
- Chlorophyll measurement: No

RANGE-FINDING TEST
An initial range-finding test was conducted at nominal loading rates of 0 (control), 1.0, 10 and 100 mg/L using solutions prepared in duplicate as Water Accommodated Fractions (WAFs). The results from the initial range-finding test showed significant inhibition of growth at all loading rates employed, so a second range-finding test was conducted at nominal concentrations of 0 (control), 0.010, 0.10 and 1.0 mg/L. Based on nominal concentrations, the results of the range-finding tests suggested that the 72-hour ExC50 value would between 0.10 and 1.0 mg/L, and definitive concentrations were selected accordingly.

The test substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB) and was considered to be of low water solubility. As such the test solutions were prepared as Water Accommodated Fractions (WAFs) for the range-finding tests as recommended by the OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures). However, during the range-finding tests, chemical analysis of the test concentrations indicated that the test substance was soluble in the aqueous OECD medium. In addition, the toxicity of the test substance was such that individual WAFs could not practically be prepared. As such, the range assigned to the definitive was based on nominal test concentrations rather than loading rates. The initial preparation method was maintained for consistency.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.9 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.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(yield)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(yield)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

(yield)

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): None reported
- Test media descriptions: The freshly prepared test solutions solutions at 0 hours appeared as colourless. The appearance of the test media following the 72-hour definitive exposure period were described as non-homogenous hazy dispersion of algae cells with color ranging from green (control) to colorless (3.2 mg/L).
- Any stimulation of growth found in any treatment: Yes
- Any observations that might cause a difference between measured and nominal values: Given that measured concentrations within the toxicity range were within 80% to 120% of nominal value, the results have been based on nominal concentrations.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

A positive control study was conducted to determine the effects of the reference substance, potassium dichromate, on algal growth by exposing the green alga, Pseudokirchneriella subcapitata, at the exponential growth phase to the reference substance at nominal concentrations of 0.1, 0.32, 1.0 and 3.2 mg/L, along with a control, for 72 hours at 21-24˚C under static conditions with continuous illumination. The 72-hour EC50 values for yield, area under the growth curve and growth rate were determined to be 1.19, 1.21 and 1.93 mg/L, respectively. The 72-hour NOEC value for yield, area under the growth curve and growth rate was determined to be 0.32 mg/L.

These results were within the expected toxicity range (growth rate EC50: 0.38 - 2.60 mg/L) for this reference substance and all validity criteria were met; the test is therefore considered valid.

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. The average specific growth rate (µ) and the final yield over specified time intervals were analysed using Dunnett Multiple Comparison test to determine the no observed effect concentration (NOEC). Linear interpolation analysis was performed in order to estimate EC10, EC20 and EC50 values. Where possible, 95% confidence limits were calculated for the EC10, EC20 and EC50 values.

Percent inhibition of growth rate of Pseudokirchneriella subcapitata 

Nominal loading rate	Average specific growth rate, “µ” (cells x 10^-2/mL)				Inhibition of average specific growth  rates relative to the control (%)
(mg/L)	0-24 hours	24-48 hours	48-72 hours	0-72 hours	0-24 hours	24-48 hours	48-72 hours	0-72 hours
Control	7.94	5.23	5.28	6.15	-	-	-	-
0.032	6.80	5.53	6.16	6.16	14	-6	-17	0
0.10	7.14	5.71	5.18	6.01	10	-9	2	2
0.32	6.88	4.81	5.79	5.83	13	8	-10	5
1.0	5.84	4.37	3.64	4.62	26	17	31	25
3.2	2.12	5.88	-3.20	1.60	73	-12	161	74

 - Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

 

 

Percent inhibition of yield of Pseudokirchneriella subcapitata 

Nominal loading rate (mg/L)	Initial cell density (cells/mL)	Yield at 72 hours * (cells/mL)	Mean Final Yield per treatment (cells/mL)	% Inhibition in Final Yield relative to control treatment
Control	1 E04	782920 833700 890420 882960 788380 796560	82.9E04	-
1.0	1 E04	853620 787420 863220	83.5E04	-1
3.2	1 E04	863040 632860 765200	75.4E04	9
10	1 E04	702460 648020 616180	65.6E04	21
32	1 E04	263300 213000 342220	27.3E04	67
100	1 E04	22060 20380 22440	2.16E04	97

* Final yield (cells/mL) = cell density (cells/mL) at 72 hours – initial cell density (cells/mL)

- Not applicable

Negative percentage inhibition values relative to the control indicate growth stimulation and are not considered an adverse effect.

# Replicate not included in results calculations due to unusual growth. Statistical analysis confirmed the replicate to be an outlier (Grubbs Extreme Value test).

Validity criteria fulfilled:

yes

Remarks:

The control cell densities increased by a factor of 83. The mean coefficient of variation (CV) for section-by-section specific growth rate in the control cultures was 7.82% and the overall CV for specific growth rate was 1.28%.

Conclusions:

Based on nominal concentrations, the 72-hour effects on growth rate (ErC50) and yield (EyC50) were determined to be 1.9 mg/L and 0.71 mg/L, respectively. The corresponding EC10 and NOEC values for specific growth rate after 72 hours were 0.46 mg/L and 0.1 mg/L, respectively.

Executive summary:

The study was conducted in accordance with the requirements of OECD Guideline No. 201, under GLP conditions to assess the effects of the test substance on the growth of the freshwater green alga, Pseudokirchneriella subcapitata, during a 72-hour growth inhibition toxicity test. Based on the results of two rangefinding test, the definitive test was conducted under static conditions at nominal loading rates of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/L. Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1 x 10^4 algae cells/mL and incubated at 21 - 24°C with continuous lighting (4440 - 8880 lux) for 72 hours. Cell counts were performed at 24-hour intervals. Water quality was measured at 0 and 72 hours.

Samples of test media were analysed at 0 (new media) and 72 hours (expired test media, pooled by replicate) usingliquid chromatography with time-of-flight tandem mass spectrometry detection (LC-TOF/MS) based on quantitation of based on quantitation of phosphoric acid dimethyl ester constituent, the marker constituent with the most singularly abundant peak. Analysis of the test preparations at 0 and 72 hours showed measured concentrations to range from 88% to 101% of nominal value with the exception of the 0.032 mg/L test concentration at 0 hours, which showed a measured concentration of 79% of nominal. This low measured concentration was considered not to have affected the integrity of the test because the 0.032 mg/L test concentration was below the No Observed Effect Concentration. Given that measured concentrations within the toxicity range were within 80% to 120% of nominal value, the results were based on nominal concentrations.

Based on nominal concentrations, the 72-hour effects on growth rate (ErC50) and yield (EyC50) were determined to be 1.9 mg/L and 0.71 mg/L, respectively. The corresponding EC10 and NOEC values for specific growth rate after 72 hours were 0.46 mg/L and 0.1 mg/L, respectively.

Description of key information

In an OECD Guideline 201 study, conducted according to GLP, the effect of the test substance on the growth of the freshwater algae, Pseudokirchneriella subcapitata, was investigated and, based on nominal concentrations, the 0-72 hour ErC50 (growth rate) and the 72-hour EyC50 (yield) values were determined to be 1.9 mg/L and 0.7 mg/L, respectively. The corresponding EC10 and NOEC values for specific growth rate after 72 hours were 0.46 mg/L and 0.1 mg/L, respectively (Smithers Viscient (ESG) Ltd, 2018). 

Key value for chemical safety assessment

EC50 for freshwater algae:

1.9 mg/L

EC10 or NOEC for freshwater algae:

0.46 mg/L