Dipentylammonium dipentyldithiocarbamate

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:

30 January 2018 to 22 November 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:

CAS RN: 71902-20-0
Purity: >95%
This substance has an 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: Samples (10 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 8 days at -20˚C. Although stability data met 80-120% criteria, a difference of 27% was seen for stored recoveries relative to freshly prepared recovery solutions. Measured concentrations determined for stored samples should therefore be viewed with caution.
- Sample processing: Prior to analysis, the samples were treated with calcium chloride solution, then mixed with acetonitrile containing 0.2% formic acid, followed by further dilution, as required, with acetonitrile / OECD medium (1:1, v/v) containing 0.1% formic acid and calcium chloride to bring the response within the calibration range.

Vehicle:

no

Details on test solutions:

The test substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB) and was expected to be of low water solubility. The test solutions were therefore 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 test, chemical analysis of the test concentrations indicated that the test substance was soluble in the test medium. As such, the definitive test solutions were prepared based on nominal test concentrations rather than WAF loading rates. The initial preparation method of the stock solution was maintained for consistency.

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 10, 3.2, 1.0, 0.32 and 0.10 mg/L test concentrations. The control solution was prepared by adding OECD medium only to the control vessels.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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:

22.6 - 26.3 °C

The protocol specified a temperature range of 21 to 24ºC, maintained at ±2ºC. This deviation was considered not to affect the integrity of the test given the control validity criteria were achieved, indicating good algal growth under the conditions employed.

pH:

7.60 - 8.32

Dissolved oxygen:

not specified

Salinity:

Not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L
Measured concentrations: 0 (control) 0.069, 0.27, 0.82, 2.6, 8.4 and 23 mg/L (equivalent to 69, 84, 82, 81, 84 and 72% of nominal, respectively)

Given that some of the measured concentrations were outside the 80% to 120% of nominal value, the results have been based on geometric mean measured concentrations.

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: 6530 – 6890 lux (0 hour); 6060 – 6440 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
- Cell condition: At the end of the test, a subsample of the pooled replicates from the control and each test concentration was taken and the shape and general condition of the algae in each test group was assessed by microscopic observations.

RANGE-FINDING TEST
A range-finding test was conducted at nominal test substance loading rates of 1.0, 10 and 100 mg/L. A concurrent control group was also included. Duplicate test vessels were prepared for the control and each test loading rate.

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: (95% confidence limits 3.4 - 12)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% confidence limits, 14 - 17)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.82 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% confidence limits, not calculable - 9.6)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% confidence limits, 8.1-10)

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 media for the control and 0.10 to 3.2 mg/L test concentrations (nominal) at 0 hours appeared as colourless solutions. The 10 and 32 mg/L test concentrations (nominal) were observed to be pale white homogenous hazy dispersions.
The appearance of the test media following the 72-hour exposure period was described as a homogenous hazy dispersion of algal cells, green to pale green at 0.069 - 0.82 mg/L, and a homogenous hazy dispersion of test substance, very pale green to pale white at 2.6 - 23 mg/L (based on mean measured 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 for 72 hours. The 72-hour EC50 values for yield, area under the growth curve and growth rate were determined to be 0.599, 0.604 and 0.875 mg/L, respectively. The 72-hour NOEC value for yield, area under the growth curve and growth rate were determined to be 0.32, <0.1 and 0.32 mg/L, respectively.

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 Bonferroni Adjusted t-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 ofPseudokirchneriella subcapitata

Geometric mean measured concentration	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	5.47	6.27	6.17	5.97	-	-	-	-
0.069	4.95	6.41	6.46	5.94	10	-2	-5	0
0.27	4.85	6.36	6.38	5.86	11	-1	-4	2
0.82	5.33	6.39	6.49	6.07	3	-2	-5	-2
2.6	5.30	6.22	5.12	5.54	3	1	17	7
8.4	4.84	6.04	4.57	5.15	12	4	26	14
23	3.77	1.14	-0.018	1.63	31	82	100	73

- Not applicable

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

 

Percent inhibition of yield ofPseudokirchneriella subcapitata

Geometric mean measured concentration (mg/L)	Initial cell density (cells x 10^4/mL)	Yield at 72 hours * (cells/mL)	Mean Final Yield per treatment (cells x 10^4/mL)	% Inhibition in Final Yield relative to control
Control	1	766900 678920 858240 706580 659280 690760	72.7	-
0.069	1	910600 670560 587500	72.3	1
0.27	1	641600 636240 740520	67.3	7
0.82	1	787920 715740 841180	78.2	-8
2.6	1	444800 570300 590320	53.5	26
8.4	1	338560 462020 400680	40.0	45
23	1	28200 20420 19300	2.26	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.

Validity criteria fulfilled:

yes

Remarks:

The control cell densities increased by a factor of 73. The mean coefficient of variation (CV) for section-by-section specific growth rate in the control cultures was 9.12% and the overall CV for specific growth rate was 2.25%.

Conclusions:

Based on geometric mean measured concentrations, the 72-hour EyC50 (yield) and the 0-72 hour ErC50 (growth rate) values were calculated to be 9.3 and 16 mg/L, respectively. The corresponding No Observed Effect Concentration (NOEC) values for yield and specific growth rate after 72 hours were 0.82 mg/L.

Executive summary:

The objective of the study was to determine the effects of the test substance on the growth of the freshwater green alga, Pseudokirchneriella subcapitata, during a 72-hour growth inhibition toxicity test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201. Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006, Annex 5 corrected 28 July 2011). The definitive test was conducted at nominal concentrations of 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L, which were selected based on the results of a range‑finding test, for which only the key results were reported. A concurrent control group was also included.

Test vessels (250 mL conical flasks) were prepared containing 100 mL of the appropriate test or control medium. Each test vessel was inoculated with 1x10^4 algae cells/mL and incubated at 21 - 24°C (under 4440-8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Samples of test media were analysed at 0 (new media) and 72 hours (old test media). Concentrations of the test substance were determined byliquid chromatography triple quadruple mass spectrometry (LC-TQMS), using the dipentylamine constituent as an analytical marker for quantification.

Analysis of the test preparations at 0 and 72 hours showed measured concentrations to range from 80% to 85% of nominal value, with the exception of the 0.10 and 32 mg/L test concentrations at 72 hours which showed measured concentrations to be 58% and 62% of nominal value, respectively. Given that some of the measured concentrations were outside the 80% to 120% of nominal value, the results have been based on geometric mean measured concentrations, which were calculated to be 0.069, 0.27, 0.82, 2.6, 8.4 and 23 mg/L (equivalent to 69, 84, 82, 81, 84 and 72% of nominal value, respectively).

The 72-hour EyC50 (yield) and the 0-72 hour ErC50 (growth rate) values were determined to be 9.3 and 16 mg/L, respectively. The No Observed Effect Concentration (NOEC) for yield and specific growth rate after 72 hours was determined to be 0.82 mg/L.

Description of key information

In an OECD 201 study, conducted according to GLP, the effect of the test substance on the growth of the freshwater green algae, Pseudokirchneriella subcapitata, was investigated and the 72-hour EyC50 (yield) and the 0-72 hour ErC50 (growth rate) values were determined to be 9.3 and 16 mg/L, respectively. The No Observed Effect Concentration (NOEC) for yield and specific growth rate after 72 hours was determined to be 0.82 mg/L (Smithers Viscient (ESG) Ltd., 2018).

.

Key value for chemical safety assessment

EC50 for freshwater algae:

16 mg/L