Fatty acids, C18-unsatd., phosphates

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:

February- June 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)

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures

Version / remarks:

OECD series on testing and assessment number 23, 2000.

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 14 September 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the glasswool used in the preparation of the highest WAF and containing undissolved residue was kept for possible analysis.

Frequency at t=0 h, t=24 h and t=72 h
Volume 2.7 mL
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 concentration before sampling.

Vehicle:

no

Details on test solutions:

Preparation of Test Solutions
The batch of Fatty acids, C18-unsatd., phosphates tested was a yellow liquid UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with loading rates individually prepared at loading rates of 1.0 to 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
Due to the observed degree of adsorption during the analytical method development, all glassware employed in this study was silanized prior to use.
Any residual volumes were discarded

Test organisms (species):

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

Details on test organisms:

Species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21-24°C, constant within 2°C

pH:

8.1 +- 0.2

Nominal and measured concentrations:

blank control, 1.0, 10 and 100 mg/l

Details on test conditions:

Stock culture 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.
Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock 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) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-culture 3 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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Merck, Art. 1.04864, Batch K44879664).

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 13 to 17 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: yield inhibition

Remarks on result:

other: 95% confidence interval ranging from 1.1 to 1.3 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: yield inhibition were 0.34 mg/L.

Details on results:

Combined Limit/Range-Finding Test
The mean cell densities measured during the combined limit/range-finding test are presented
in Table 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield
inhibition per concentration, respectively.
A dose-related increase of inhibition of both growth rate and yield was observed at the end of
the test, resulting in 64% and 97% inhibition of growth rate and yield, respectively.
Samples taken from WAFs prepared at loading rates of 1.0 and 100 mg/L were analysed
employing two out of several peaks observed in the chromatograms of the test item. The
initial concentrations based on the peak at m/z 151.0 were 1.1 and 106 mg/L, respectively,
and remained stable throughout the test duration, i.e. were at 108-115% of the initial
concentrations at the end of the test (see also Table 3 of the appended Analytical Report).
Based on the peak at m/z 391.3, the initial concentrations were 0.93 and 95 mg/L,
respectively. The higher concentration remained stable and was at 102% of the initial
concentration at the end of the test. The lower concentration decreased below the lowest
calibration standard used in the chemical analysis of the samples (see also Table 4 of the
appended Analytical Report).
All test conditions were maintained within the limits prescribed by the study plan.

Final Test
The results of analysis of the samples taken during the final test are described in Table 5 and
Table 6 of the appended Analytical Report.
Samples taken from all test concentrations were analysed were analysed employing two out
of several peaks observed in the chromatograms of the test item.
Based on the peak at m/z 151.0, the concentrations at the start of the test were 1.1, 3.5, 10, 34
and 98 mg/L in WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L,
respectively. The measured concentrations remained stable throughout the test duration, i.e
were at 102-108% of the initial concentrations at the end of the test.
Based on the peak at m/z 391.3, the concentrations at the start of the test were 0.48, 1.8, 6.8,
32 and 93 mg/L in WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L,
respectively. At the end of the test, the two lowest test concentrations had decreased below
the lowest calibration standard used in the chemical analysis of the samples, while the WAFs
prepared at loading rates of 10 and 30 mg/L had decreased to 1.1 and 31% of the initial
concentration, respectively. The highest test concentration remained stable throughout the test
duration, i.e. was at 96% of the initial concentrations at the end of the test.
The concentrations in the samples without algae were comparable to the according samples
with algae based on either peak.
Following the worst-case assumption, the effect parameters were based on the results
obtained for the peak at m/z 391.3. To this end, the Time Weighted Average (TWA)
concentrations were calculated for the peak at m/z 391.3 (see Table 4) and used to determine
the effect parameters.

Mean Cell Densities
Figure 1 shows growth curves at different concentrations of Fatty acids, C18-unsatd.,
phosphates. The individual and group mean cell densities measured at 24h intervals are given
in Table 10.

Inhibition of Growth Rate and Inhibition of Yield
Table 5 shows group mean growth rates and the percentages of growth rate inhibition (total
test period), whereas Table 6 shows the values at different time intervals. The group mean
yields and the percentages of yield inhibition are summarized in Table 7 (see Appendix 1 for
the individual values). Statistical analysis of the data is shown in Appendix 2 and Appendix 3.
Growth rates were in the range of the controls at the concentrations 0.34 mg/L and below
during the 72-hour test period, whereas the growth rate of algae exposed to 1.2 and 22 mg/L
were increasingly reduced. At the highest concentration tested, inhibition of algal growth
stagnated. Statistically significant inhibition of growth rate was found at TWA concentrations
of 1.2 mg/L and higher.
Inhibition of yield was statistically significant at the three highest test concentrations.
Microscopic observations at the end of the test revealed a normal and healthy appearance of
the algal cells exposed to all test concentrations when compared to the control.

Experimental Conditions
Table 9 shows the pH recorded at the beginning and the end of the test. The pH was within
the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5
units). During the exposure period the temperature measured in the incubator was maintained
between 21 and 23°C. Temperature remained within the limits prescribed by the study plan
(21-24°C, constant within 2°C).

Table 1
Mean Cell Densities (x10⁴Cells/mL) During the Combined Limit/Range-Finding Test

Time (h)	Fatty acids, C18-unsatd., phosphates WAF loading rate (mg/L)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	5.8	n.d.	n.d.	3.2
48	34.3	n.d.	n.d.	5.0
72	169.5	170.7	93.6	6.3

n.d.: not determined

Table 2

Percentage Inhibition of Growth Rate During the Combined Limit/Range-Finding Test

Fatty acids, C18-unsatd., phosphates WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.710	0.0213	6
1.0	1.712	0.0406	3	-0.07
10	1.512	0.0272	3	12
100	0.611	0.0261	6	64

 

Table3
Percentage Inhibition of Yield During the Combined Limit/Range-Finding Test

Fatty acids, C18-unsatd., phosphates WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	168.5	11.04	6
1.0	169.7	20.25	3	-0.68
10	92.6	7.56	3	45
100	5.3	0.50	6	97

Table 4
Measured Concentrations Versus Nominal Concentrations(m/z391.3)

Fatty acids, C18-unsatd., phosphates WAF loading rate (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
1.0	0.475	0.0618	0.0039^2	0.067
3.2	1.81	0.418	0.0039^2	0.32
10	6.79	1.24	0.012^2	1.0
10^1	7.13	1.73	0.076	1.4
32	32.1	29.5	0.0882	11
100	93.0	96.3	9.98	52

¹without algae;²estimated as being a factor 2 below the lowest calibration standard.

 

   

Table 4
Measured Concentrations Versus Nominal Concentrations(m/z391.3)

Fatty acids, C18-unsatd., phosphates WAF loading rate (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
1.0	0.475	0.0618	0.0039^2	0.067
3.2	1.81	0.418	0.0039^2	0.32
10	6.79	1.24	0.012^2	1.0
10^1	7.13	1.73	0.076	1.4
32	32.1	29.5	0.0882	11
100	93.0	96.3	9.98	52

¹without algae;²estimated as being a factor 2 below the lowest calibration standard

Table5
Growth Rate And Percentage Inhibition For The Total Test Period

Fatty acids, C18-unsatd., phosphates TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.544	0.0251	6
0.067	1.523	0.0227	3	1.4
0.34	1.567	0.0165	3	-1.5
1.2	1.302	0.0223	3	16*
22	0.487	0.0413	3	68*
93	0.548	0.0662	3	64*

* effect was statistically significant

Table 6
Growth Rate And Percentage Inhibition At Different Time Intervals

Fatty acids, C18-unsatd., phosphates TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.264		1.495		1.873
0.067	3	1.042	18	1.623	-8.6	1.903	-1.6
0.34	3	1.267	-0.21	1.58	-5.7	1.853	1.1
1.2	3	1.314	-4.0	0.929	38	1.663	11
22	3	0.762	40	1.026	31	-0.327	117
93	3	0.598	53	0.937	37	0.11	94

Table 7
Yield And Percentage Inhibition For The Total Test Period

Fatty acids, C18-unsatd., phosphates TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	102.0	7.64	6
0.067	95.6	6.68	3	6.3
0.34	109.0	5.41	3	-6.9
1.2	48.8	3.27	3	52*
22	3.3	0.52	3	97*
93	4.3	1.09	3	96*

* effect was statistically significant

Table8
Effect Parameters

		Parameter (mg/L)		NOEC		EC10		EC20		EC50
Growth rate		Value		0.34		0.53		1.7		15
		lower 95%-cl				0.41		1.4		13
		upper 95%-cl				0.66		1.9		17
Yield		Value		0.34		0.44		0.62		1.2
		lower 95%-cl				0.39		0.57		1.1
		upper 95%-cl				0.48		0.66		1.3

cl: confidence limit

 

Table 9
pH Levels Recorded During the Final Test

Fatty acids, C18-unsatd., phosphates TWA conc. (mg/L)	pH
	t=0h	t=72h
Control	8.2	8.1
0.067	8.1	8.1
0.32	8.1	8.1
1.0	8.1	8.0
11	8.0	7.9
52	7.8	7.8

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata
(formerly known as Pseudokirchneriella subcapitata), Fatty acids, C18-unsatd., phosphates
reduced growth rate and inhibited the yield of this fresh water algae species significantly at
TWA concentrations of 1.2 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 15 mg/L with a 95% confidence interval
ranging from 13 to 17 mg/L.
The EC50 for yield inhibition (72h-EYC50) was 1.2 mg/L with a 95% confidence interval
ranging from 1.1 to 1.3 mg/L.
The 72h-NOEC for growth rate inhibition and the 72h-NOEC for yield inhibition were
0.34 mg/L.

Executive summary:

The objective of the study was to evaluate Fatty acids, C18-unsatd., phosphates for its ability

to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella

subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC,

EC10 and EC50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201,

2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test

methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Fatty acids, C18-unsatd., phosphates tested was a yellow liquid UVCB and not

completely soluble in test medium at the loading rates initially prepared. Due to the observed

degree of adsorption during the analytical method development, all glass ware employed in

this study was silanized prior to use.

Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0 to

100 mg/L and used as test concentrations.

A final test was performed based on the results of a preceding combined limit/range-finding

test. Six exponentially growing algal cultures were exposed to an untreated control, whereas

three replicates per group were exposed to WAFs prepared at loading rates of 1.0, 3.2, 10, 32

and 100 mg Fatty acids, C18-unsatd., phosphates per litre. The initial algal cell density was

104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation

of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Algal growth was in the range of the controls at the two lowest test concentrations during the

72-hour test period, whereas inhibition of growth rate and yield at the three highest test

concentrations was statistically significant.

Samples taken from all test concentrations were analysed employing two out of several peaks

observed in the chromatograms of the test item. Based on the peak at m/z 151.0, the

concentrations at the start of the test were 1.1, 3.5, 10, 34 and 98 mg/L in WAFs prepared at

loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, respectively. The measured concentrations

remained stable throughout the test duration, i.e were at 102-108% of the initial

concentrations at the end of the test.

Based on the peak at m/z 391.3, the concentrations at the start of the test were 0.48, 1.8, 6.8,

32 and 93 mg/L in WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L,

respectively. At the end of the test, the two lowest test concentrations had decreased below

the lowest calibration standard used in the chemical analysis of the samples, while the WAFs

prepared at loading rates of 10 and 30 mg/L had decreased to 1.1 and 31% of the initial

concentration, respectively. The Highest test concentration remained stable throughout the

test duration, i.e. was at 96% of the initial concentrations at the end of the test.

Following the worst-case approach, the effect parameters were based on the results obtained

for the peak at m/z 391.3. To this end, the Time Weighted Average (TWA) concentrations

were calculated for the peak at m/z 391.3 (i.e. 0.067, 0.34, 1.2, 22 and 93 mg/L in WAFs

prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, respectively) and used to

determine the effect parameters.

The study met the acceptability criteria prescribed by the study plan and was considered

valid.

Description of key information

The 72-hour toxicity of algae (Raphidocelis subcapitas) to the registered substance was determined according to OECD 201 guideline in compliance with GLP. The study was conducted based on nominal concentrations. Nominal concentrations of 1.1, 3.5, 10, 34 and 98 mg/L were used. As a result, EC50 and NOEC values were determined basis for effect yield inhibition and growth rate. Therefore, based on the available data, the lowest value (EC50) was selected as a key value.

Key value for chemical safety assessment

EC50 for freshwater algae:

13 mg/L

EC50 for marine water algae:

13 mg/L

EC10 or NOEC for freshwater algae:

0.34 mg/L

EC10 or NOEC for marine water algae:

0.34 mg/L