3-(heptyloxy)propane-1,2-diol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Sep 2019 - 22 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-octoxypropane-1,2-diol

Cas Number:

10438-94-5

Molecular formula:

C11H24O3

IUPAC Name:

3-octoxypropane-1,2-diol

Test material form:

liquid

Details on test material:

Storage Conditions: At room temperature protected from light container flushed with nitrogen.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h, t=24 h and t=72 h.
Volume: 1.5 mL from the approximate centre of the test vessels.
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were pooled at each test group before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a test item concentration of nominally 10 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 1.5 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Method: The test item was completely soluble in test medium at the concentrations tested based on visual inspection. Test item was dosed to test medium on a volume basis. The required volume was calculated using the available density of the test item (0.964 g/mL) as provided by the Sponsor. Preparation of test solutions started with the highest concentration of 100 mg/L applying 15 minutes of magnetic stirring to accelerate dissolution of the test item in medium and assure homogeneous solutions. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
Purity correction: No correction was made for the purity/composition of the test item.
Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.
Preparation conditions: Due to the light sensitivity of the test item, preparation of test solutions was performed under dimmed light conditions and glassware used was wrapped in aluminium foil.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Age of inoculum (at test initiation): Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

between 23 and 24°C

pH:

7.7 - 8.1

Dissolved oxygen:

not applicable

Salinity:

not applicable

Conductivity:

not aplicable

Nominal and measured concentrations:

Nominal concentrations: Nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L and an untreated control.
Measured concentrations: The measured concentrations at the start of the test were 1.2, 3.9, 14, 38, and 131 mg/L at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L, respectively. The concentrations were in the range of 70 to 94% relative to the initial values at the end of the test. No test item was detected in the control samples taken throughout the test.Based on the obtained results, the Time Weighted Average (TWA) concentrations were calculatedto be 1.1, 3.6, 12, 38, and 128, corresponding with 1.0, 3.2, 10, 32 and 100 mg/L, respectively.
The initial concentration measured in the solution incubated without algae (abiotic control) remained stable throughout the test, while the concentration in the solution with algae decreased slightly over time. Hence, the presence of algae affected the concentration of the test item in test medium.

Details on test conditions:

TEST SYSTEM
- Test vessel:100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: A mean cell density in the control at the end of the test of 270 * 10^4 cells/mL
- No. of vessel per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Culture medium: M1: according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

TEST MEDIUM / WATER PARAMETERS
- Test medium: M2: according to the OECD 201 Guideline
- Culture medium different from test medium: Yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity within the range of 74 to 79 µE.m^-2.s^-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations:At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank.
- Effect parameters:72 h NOECrC, 72 h NOEyC, 72 h ErC50, 72 EyC50, 72 h ErC20, 72 h EyC20, 72 h ErC10 and 72 h EyC10.

TEST CONCENTRATIONS
Combimed limit/rang-finding test:
- Test concentrations: exponentially growing algal cultures were exposed to a range of nominally 0.10 to 100 mg/L increasing by a factor of 10 and to a blank control.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

34 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 28 - 40 mg/L

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

87 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Confidence interval: 80 - 95 mg/L

Details on results:

- Exponential growth in the control: Yes
- Obvervations of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 38 mg/L when compared to the control.

Results with reference substance (positive control):

The 72h-EC50 for growth rate inhibition (ERC50) was 2.02 mg/L with a 95% confidence interval ranging from 2.00 to 2.04 mg/L.
In conclusion, the sensitivity of this culture of Raphidocelis subcapitata was in agreement with the historical data collected at Charles River Den Bosch.

Reported statistics and error estimates:

For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (step-down Jonckheere-Terpstra Test, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Table 1: 
Measured Concentrations in the Final Test and Calculation of Time Weighted Average Concentrations

Saskine^TM80 Nominal conc. (mg/L)	Measured concentration (mg/L)			TWA conc. (mg/L)
	t=0h	t=24h	t=72 h
1.0	1.21	1.24	0.876	1.1
3.2	3.89	4.23	2.73	3.6
10	13.6	12.5	9.53	12
10*	12.3*	11.8*	12.1*	12*
32	38.1	41.5	34.2	38
100	131	130	123	128

* abiotic control, incubated without algae.

Table 2:         
Growth Rate and Percentage Inhibition for the Total Test Period

Saskine^TM80 Nominal concentration (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.865	0.0222	6
1.0 [1.1]	1.863	0.0298	3	0.14
3.2 [3.6]	1.884	0.0056	3	-1.0
10 [12]	1.857	0.0179	3	0.47
32 [38]	1.624*	0.0256	3	13
100 [128]	0.560*	0.1729	3	70

* Statistically different from the control, [ ] – TWA concentration (mg/L).

Table 3:

Growth Rate and Percentage Inhibition at Different Time Intervals 

Saskine^TM80 Nominal concentration (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72 h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.078		1.901		1.617
1.0 [1.1]	3	1.959	5.7	1.930	-1.5	1.699	-5.1
3.2 [3.6]	3	2.079	-0.075	1.922	-1.1	1.651	-2.1
10 [12]	3	1.989	4.3	1.899	0.092	1.682	-4.0
32 [38]	3	1.751	16	1.743	8.3	1.378	15
100 [128]	3	0.811	61	0.475	75	0.395	76

[ ] – TWA concentration (mg/L).

Table 4:

Effect Parameters

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	12	34	47	87
	lower 95%-cl		28	40	80
	upper 95%-cl		40	53	95
Yield	Value	12	17	22	37
	lower 95%-cl		12	17	34
	upper 95%-cl		20	25	40

cl – confidence limit.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see "overal remarks"

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, Saskine TM 80 inhibited growth rate and yield of this freshwater algae species significantly at TWA concentrations of 38 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 87 mg/L with a 95% confidence interval ranging from 80 to 95 mg/L.

Executive summary:

A 72 hour toxicity study with freshwater algae (pseudokirchneriella subcapitata) was conducted according tot OECD guideline 201 and GLP principles. A final test was performed based on the results of a range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The initial algal cell density was 10^4cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure. The Time Weighted Average (TWA) concentrations were calculated to be 1.1, 3.6, 12, 38 and 128 mg/L at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L, respectively. No statistically significant growth rate inhibition was recorded at TWA concentrations up to and including 12 mg/L. Statistically significant growth rate inhibition of 13% and 70% was found at TWA concentrations of 38 and 128 mg/L, respectively.

The 72h-EC₁₀ for growth rate inhibition (E_RC₁₀) was 34 mg/L with a 95% confidence interval ranging from 28 to 40 mg/L. The 72h-EC₅₀for growth rate inhibition (E_RC₅₀) was 87 mg/L with a 95% confidence interval ranging from 80 to 95 mg/L.

The study met the accepability criteria, and is considered to be valid.