Hexadecan- l-ol, ethoxylated

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Aug to 26 Aug 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 100 mg/L
- Sampling method: samples taken after test preparation at 0 hours and from the pooled replicates at 72 hours, and at 24h and 48 h
- Sample storage conditions before analysis: All 0, 24 and 48-Hour samples were stored frozen prior to analysis alongside the 72-Hour test samples.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (200 mg) was weighed onto a glass slide and suspended under the surface of 2 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1-Hour. Visual observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 100 mL discarded). Further filtration through a single sheet of coarse filter paper was conducted to remove as much undissolved test item as possible. Microscopic observations of the WAF were performed after filtering and showed there to be no micro dispersions of test item present.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ±1 ºC

pH:

7.7 - 8.8

Nominal and measured concentrations:

Nominal test substance concentration: 100 mg/L (WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): plugged with polyurethane foam bungs
- Fill volume: 100 mL
- Initial cells density: 5.00 x 10*3 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux, warm white lighting (380 to 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicaple
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no effect on growth at 1.0, 10 and 100 mg/L loading rate WAF

CULTURING APPARATUS
-Details on culturing apparatus used: INFORS Multitron (R) incubator

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes

Analysis of the 100 mg/L loading rate WAF test preparation at 0 hours showed a measured test concentration of 0.015 mg/L was obtained. Analysis at 24, 48 and 72 hours showed a decline in measured test concentration occurred to less than the LOQ of the analytical method employed, determined to be 0.00053 mg/L. Analysis of a 100 mg/L loading rate WAF sample prepared at the start of the test to which no algal cells were added also showed a measured test concentration of less than the LOQ after 72 hours exposure indicating that the decline observed was due to instability of the test item under test conditions rather than adsorption of the test item to the algal cells present.

Results with reference substance (positive control):

ErC50 (0 to 72 hour): 1.2 mg/L; 95% confidence limits 1.1 to 1.3 mg/L

Reported statistics and error estimates:

All statistical analyses were performed using the ToxRatPro software package Version 3.2.1 (TOXRAT, 2001-2015).

Table 1 Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.077	-	1.29E+06	-
	R2	0.076		1.22E+06
	R3	0.076		1.21E+06
	R4	0.079		1.50E+06
	R5	0.077		1.23E+06
	R6	0.075		1.14E+06
	Mean	0.077		1.27E+06
	SD	0.001		1.27E+05
100	R1	0.078	[1] 0 [1] [3] [3] [1] [2]	1.34E+06	[9]
	R2	0.077		1.28E+06
	R3	0.078		1.36E+06
	R1	0.079		1.49E+06
	R2	0.079		1.46E+06
	R3	0.078		1.37E+06
	Mean	0.078		1.38E+06
	SD	0.001		7.74E+04

*        In accordance with the OECD test guideline only the mean value for yield is calculated

R      = Replicate

SD   = Standard deviation

[ ]          = Increase in growth compared to controls

Table 2: Analytical dose verification

Time [h]	Nominal loading rate [mg/l]	Measured [mg/l]	% of nominal
0	Control	< LOQ	-
0	100	0.0152	-
procedural recovery1	0.00530	0.00497	94
procedural recovery2	0.00530	0.00511	96
24	100	< LOQ	-
48	100	< LOQ	-
72	Control	< LOQ	-
	100	< LOQ	-
	100 no algae	< LOQ	-

Procedural recovery: to demonstrate the validity of the analytical procedure, duplicate volumes of test medium were spiked with the test item at the required concentrations and treated as per the test samples. The concentrations quoted for the procedural recovery samples are the actual fortified concentrations. The corresponding percentage of nominal concentration values relate the Percentage of Fortified Concentration recovered.

Table 3: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	254	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	14%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1%	yes

Validity criteria fulfilled:

yes

Remarks:

Please refer to table 3 at "Any other information on results incl. tables"

Additional information

The toxicity of Hexadecan-1-ol, ethoxylated to aquatic algae was investigated in a study conducted according to OECD guideline 201. The study assessed the effect of a limit concentration of 100 mg/L (nominal loading rate, WAF) on the growth of Raphidocelis subcapitata. The test concentration was analytically monitored by HPLC/MS-MS. Based on the algal growth an ErL50 (72h): >100 mg/L (nominal, WAF) was determined (ErL10 (72h): >100 mg/L (nominal, WAF).

Studies investigating the acute and chronic toxicity of the category substance Alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO (CAS No. 68439-49-6) are still ongoing. The outcome of the studies is expected to have an impact on the evaluation of the aquatic toxicity of Hexadecan-1-ol, ethoxylated.

Therefore, the current assessment of the aquatic toxicity of the target substance Hexadecan-1-ol, ethoxylated is considered preliminary and will be updated as soon as the final study reports are available.