9-Octadecenamide, N-[(1S,2S,3R)-2,3-dihydroxy-1-(hydroxymethyl)heptadecyl]-, (9Z)-

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:

2017-10-09 to 2017-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ceramide IIIB

IUPAC Name:

Ceramide IIIB

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

no

Remarks:

No analytical monitoring was carried out due to the low solubility of the test item.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble and multi-component test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents.
Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture).
Exposure are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction
One water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L. The loading levels are based on the results of a preliminary range finding test. An appropriate amount of the test item was weighed out and transferred into a measuring flask with an appropriate amount of the test media (see Table 1 and Table 2). This dispersion was shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour at room temperature, the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fraction (WAF) was used in a limit test.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata HINDÁK, 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): four days old preculture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 h per day. Nutrient medium Z according to LÜTTGE et al. (1994)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

river water total hardness [mg CO3/l]: 80.1

Test temperature:

22.0 (21.5 - 22.5) °C

pH:

start: 7.93 (test 100 mg/l) 7.98 (control); end: 8.66 (test 100 mg/l) 8.80 (control)

Dissolved oxygen:

8.81 (river water)

Conductivity:

266 µS/m (river water)

Nominal and measured concentrations:

100 mg/L loading rate water accomodated fraction

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, provided with cotton wool plugs. The test container were pretreated with the appropriate test solutions for 72 hours under test conditions. Before the start of the exposure phase, the test container were emptied and refilled with freshly prepared test solutions.
- Fill volume: 100 mL
- Initial cells density: nominal: approximately 5 x 10 E03 E04 cells/mL; current: 6839 cells/mL
- Control end cells density: 1168024 (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no, natural river water, for details see section "any other information on materials and methods"

TEST MEDIUM / WATER PARAMETERS
river water as specified in section "any other information on materials and methods"

OTHER TEST CONDITIONS
- Sterile test conditions: The natural river water was stored at 15 ± 2 °C and at - 18± 2 °C for at least 24 hours. Freezing was found to be suitable to minimize the content of vital natural algae cells of the water as well as to reduce microbial (bacterial) activity
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: 4780 - 5720 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm

TEST CONCENTRATIONS
- Spacing factor for test concentrations: limit test
Range finding study
- Test concentrations: 1.00 - 10.0 - 100 mg/L nominal
- Results used to determine the conditions for the definitive study:
Growth Rate Inhibition: 100: 4%, 10.0: 6%, 1.00: 2% each concentration mg/l nominal
Yield Inhibition: 100: 16%, 10.0: 25%, 1.00: 9% each concentration mg/l nominal

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item loadings

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal test item leadings

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.

Results with reference substance (positive control):

- Results with reference substance valid?
- ErC50: 0.559 (95 % c.l. 0.538 - 0.581mg/L); valid range: 0.754 ± 0.518
- EyC50: 0.320 (95 % c.l. 0.242 - 0.341), valid range: 0.398 ± 0.291
- Other: tested from 2017-04-18 to 2017-04-21

Reported statistics and error estimates:

Statistical Significance (NOEC/LOEC) – Growth Rate
t-test
Normality Test (Shapiro-Wilk) Passed(P = 0.935)
Equal Variance Test: Passed (P = 0.156)

Group Name N Missing Mean Std Dev SEM
Control 6 0 1.701 0.101 0.0412
100 mg/L 6 0 1.708 0.0548 0.0224

Difference -0.00636

t = -0.136 with 10 degrees of freedom. (P = 0.895)

95 percent confidence interval for difference of means: -0.111 to 0.0981
The difference in the mean values of the two groups is not great enough to reject the possibility that the difference is due to random sampling variability. There is not a statistically significant difference between the input groups (P = 0.895).
Power of performed test with alpha = 0.050: 0.050
The power of the performed test (0.050) is below the desired power of 0.800.
Less than desired power indicates you are less likely to detect a difference when one actually exists. Negative results should be interpreted cautiously.

The program states no significant difference for the nominal test item loading 100 mg/L compared to the control.

Any other information on results incl. tables

Cell Densities

Nominal test item loading [mg/L]	Replicate	Cell density [cells/mL]
	No.	0 hours	24 hours	48 hours	72 hours
100	1	6839	26680	241048	1304824
	2	6839	21609	214995	1117502
	3	6839	23857	214421	930852
	4	6839	23277	208148	1066063
	5	6839	26206	224990	1068755
	6	6839	27543	249077	1477575
	Mean	6839	24862	225447	1160929
Control	1	6839	28957	213777	1370016
	2	6839	27037	197743	1090173
	3	6839	25045	178045	726588
	4	6839	28181	260570	1342043
	5	6839	25282	220215	868733
	6	6839	25650	226371	1610591
	Mean	6839	26692	216120	1168024

Evaluation after 72 hours

           Statistically significant differences of growth rates and yield compared to
           control values are marked (+), not significant differences are marked (-).

Nominal test item loading [mg/L]	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.75	-3		1297985	-12
	2		1.70	0		1110663	4
	3		1.64	4		924013	20
	4		1.68	1		1059224	9
	5		1.68	1		1061916	9
	6		1.79	-5		1470736	-27
	Mean	(-)	1.71	0	(-)	1154090	1
Control	1		1.77			1363177
	2		1.69			1083334
	3		1.56			719749
	4		1.76			1335204
	5		1.61			861894
	6		1.82			1603752
	Mean		1.70			1161185

Negative values = growth stimulation

Section-by-Section and Average Specific Growth Rates of the Control Group

           (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.44	2.00	1.86	1.77	0.289	16.4	22.5
	2	1.38	1.99	1.71	1.69	0.308	18.2
	3	1.30	1.96	1.41	1.56	0.356	22.9
	4	1.42	2.22	1.64	1.76	0.417	23.7
	5	1.31	2.17	1.37	1.62	0.477	29.6
	6	1.32	2.18	1.96	1.82	0.445	24.4
				Mean	1.70
				SD ±	0.10
				CV [%]	5.93

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

72 h NOEL of Ceramide IIIb to the green alga Pseudokirchneriella subcapitata based on growth rate and yield inhibition are 100 mg/l water accomodated fraction (nominal).
72h LOEL, ErL10, ErL50 based on growth rate and yield inhibition are > 100mg/l WAF (nominal).

Executive summary:

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of Pseudokirchneriella subcapitata HINDÁK, SAG 61.81 were exposed to Ceramide IIIB at a nominal limit concentrations of 100 mg/L water accommodated fraction in natural river water under static conditions. The concentration was not measured due to low water solubility.

There was no statistically significant growth inhibition in the treated algal culture as compared to the control.

NOEL (based on growth rate) and NOEL based on yield inhibition were 100 mg/L WAF (nominal).

LOEL, ErL10, ErL50 based on growth rate or yield inhibition are > 100mg/L WAF (nominal).

Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for the algal growth inhibition test in Pseudokirchneriella subcapitata.

 

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81 

Test Type: Static

Exposition: water accommodated fraction

 

72h NOEL (based on growth rate and yield inhibition): 100 mg/L WAF (nominal).

72h LOEL, ErL10, ErL50 (based on growth rate or yield inhibition): > 100mg/L WAF (nominal).