Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates

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:

27 Feb 2012 - 5 Apr 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples:
Samples of the control and each loading rate WAF test group prepared with the omission of algal cells were taken for analysis at 0 and 72 hours.
Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

WAF procedure, for details see "Any other information on materials and methods".

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae
and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4-10^5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable.

Hardness:

Not applicable.

Test temperature:

24 ± 1 °C

pH:

7.6-8.3

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentration: 6.25, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed (plugged with polyurethane foam bungs to reduce evaporation)
- Fill volume: 100 ml
- Aeration: no
- Initial cells density: 5 x 10^3 cells/ml
- Control end cells density: 8.23 E+05 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

CULTURE MEDIUM:
NaN03: 25.5 mg/L
MgCl2*6H20: 12.164 mg/L
CaCl2*H20: 4.41 mg/L
MgS04*7H20: 14.7 mg/L
K2HP04: 1.044 mg/L
NaHC03: 15.0 mg/L
H3B03: 0.1855 mg/L
MnCl2*4H20: 0.415 mg/L
ZnCl2: 0.00327 mg/L
FeCl2*6H20: 0.159 mg/L
CoCl2*6H20: 0.00143 mg/L
Na2MoO4*2H20: 0.00726 mg/L
CuCl2*2H20: 0.000012 mg/L
Na2EDTA*2H2: 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCL
* Elga Optima 15+ or Elga Purelab Option R-15 BP

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 - 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations, range finding study: 10 and 100 mg/L nominal loading
- Test concentrations, definitive study: 6.25, 12.5, 25, 50, and 100 mg/L

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.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Inhibition of Growth Rate:
There were no statistically significant differences (P≥0.05), between the control, 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAF test groups and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/L loading rate W AF.

Inhibition of Yield:
EyL10 (0- 72 h): 66 mg/L loading rate W AF
EyL20 (0 - 72 h): 89 mg/L loading rate W AF
EyL50 (0- 72 h): > 100 mg/L loading rate W AF
where EyLx is the loading rate that reduced yield by x%.
There were no statistically significant differences between the control, 6.25, 12.5, 25, 50 and 100 mg/L loading rate WAFs (P≥0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/L loading rate WAF.

Observations on Cultures:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

pH values:
The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the Iimits given in the Test Guidelines.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions anddata evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 - 72 h): 1.4 mg/L; 95% confidence limits 1.2 - 1. 7 mg/L
EyC50 (0 - 72 h): 0.59 mg/L, 95% confidence limits 0.53 - 0.65 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ELx values were determined by inspection of the growth rate and yield data after 72 hours.

Statistical Analysis
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all loading rate WAFs to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999- 2001).

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

Table 1: Cell Densities and Percentage Inhibition of Growth from the Range-tinding Test

Nominal Loading Rate (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.11E+03	1.29E+06	-	-
	R2	4.66E+03	8.03E+05
	Mean	4.88E+03	1.05E+06
10	R1	5.05E+03	6.18E+05	9	33
	R2	5.06E+03	7.80E+05
	Mean	5.05E+03	6.99E+05
100	R1	3.91E+03	3.10E+03	111	100
	R2	3.09E+03	8.32E+02
	Mean	3.50E+03	1.97E+03

The results showed no significant effect on growth rate at 10 mg/L loading rate WAF. However, growth was observed to be reduced at 100 mg/L loading rate WAF.

Based on this information loading rates of 6.25, 12.5, 25, 50 and 100 mg/L, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.

Definitive Test

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2.

Table 2: Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate (mg/L)		pH	Cell Densities* (cells per mL)		Inhibition Values (%)		pH
		0 h	0 Hours	24 Hours	47 Hours	72 Hours	72 h
Control	R1	8.1	8.40E+03	5.06E+04	2.79E+05	1.14E+06	8.3
	R2		4.78E+03	3.74E+04	1.71E+05	8.66E+05
	R3		4.87E+03	3.10E+04	1.21E+05	6.43E+05
	R4		4.66E+03	3.08E+04	1.32E+05	6.97E+05
	R5		5.86E+03	3.09E+04	1.72E+05	7.60E+05
	R6		5.10E+03	2.60E+04	1.65E+05	8.33E+05
	Mean		5.61E+03	3.44E+04	1.73E+05	8.23E+05
6.25	R1	8.0	8.47E+03	6.93E+04	3.13E+05	1.25E+06	8.3
	R2		7.85E+03	7.61E+04	3.52E+05	1.28E+06
	R3		3.19E+03	2.69E+04	1.47E+05	6.51E+05
	Mean		6.50E+03	5.74E+04	2.71E+05	1.06E+06
12.5	R1	7.8	6.35E+03	5.63E+04	2.41E+05	1.12E+06	8.2
	R2		4.38E+03	3.37E+04	1.65E+05	7.30E+05
	R3		3.60E+03	3.57E+04	1.90E+05	7.33E+05
	Mean		4.78E+03	4.19E+04	1.99E+05	8.62E+05
25	R1	7.8	7.60E+03	5.58E+04	2.50E+05	1.35E+06	8.2
	R2		4.03E+03	3.45E+04	1.64E+05	7.47E+05
	R3		4.17E+03	3.28E+04	1.49E+05	6.34E+05
	Mean		5.27E+03	4.11E+04	1.88E+05	9.11E+05
50	R1	7.7	6.43E+03	5.37E+04	1.98E+05	7.63E+05	8.2
	R2		4.58E+03	3.68E+04	1.44E+05	6.74E+05
	R3		3.80E+03	3.29E+04	1.41E+05	6.69E+05
	Mean		4.94E+03	4.11E+04	1.61E+05	7.02E+05
100	R1	7.6	4.85E+03	4.42E+04	2.08E+05	8.23E+05	8.1
	R2		4.59E+03	4.15E+04	1.59E+05	6.04E+05
	R3		5.33E+03	3.60E+04	1.61E+05	5.65E+05
	Mean		4.92E+03	4.05E+04	1.76E+05	6.64E+05

Daily specific growth rates for the control cultures are given in Table 3.

Table 3: Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Nominal Loading Rate (mg/L)		Daily Specific Growth Rate (cells/mL/hour)
		Day 0-1	Day 1-2	Day 2-3
Control	R1	0.096	0.074	0.056
	R2	0.084	0.066	0.056
	R3	0.076	0.059	0.067
	R4	0.076	0.063	0.067
	R5	0.076	0.075	0.060
	R6	0.069	0.080	0.065
	Mean	0.080	0.070	0.063

Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

Table 4: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0-72 h	% Inhibition	0-72 h	% Inhibition*
Control	R1	0.075	-	1.13E+05	-
	R2	0.072		8.61E+05
	R3	0.067		6.39E+05
	R4	0.069		6.92E+05
	R5	0.070		7.55E+05
	R6	0.071		8.28E+05
	Mean	0.071		8.18E+05
	SD	0.003		1.74E+05
6.25	R1	0.077	[8]	1.24E+06
	R2	0.077	[8]	1.28E+06
	R3	0.068	4	6.48E+05
	Mean	0.074	[4]	1.06E+06
	SD	0.005		3.54E+05
12.5	R1	0.075	[6]	1.12E+06
	R2	0.069	3	7.26E+05
	R3	0.069	3	7.29E+05
	Mean	0.071	0	8.57E+05
	SD	0.003		2.25E+05
25	R1	0.078	[10]	1.34E+05
	R2	0.070	1	7.43E+05
	R3	0.067	6	6.30E+05
	Mean	0.072	[1]	9.06E+05	[11]
	SD	0.006		3.84E+05
50	R1	0.070	1	7.57E+06
	R2	0.068	4	6.70E+05
	R3	0.068	4	6.66E+05
	Mean	0.069	3	6.97E+05	15
	SD	0.001		5.14E+04
100	R1	0.071	0	8.18E+05
	R2	0.067	6	6.00E+05
	R3	0.066	7	5.60E+05
	Mean	0.068	4	6.59E+05	19
	SD	0.003		1.39E+05

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

SD = Standard Deviation

[Increase in growth as compared to controls]

The mean cell densities versus time for the definitive test are presented in Figure 1.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 147 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours:

- Mean cell density of control at 0 hours: 5.61 x 10 3 cells per mL

- Mean cell density of control at 72 hours 8.23 x 10 5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 15% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth Data

From the data given in Table 2 and Table 3, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence ofthe test item over the 72-Hour exposure period. It was considered that the significant inhibition of growth observed at 100 mg/1 loading rate W AF in the range-finding test was due to possible contamination of the WAF preparation. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Observations on Test ltem Solubility

Observations on the test media were carried out during the mixing and testing of the WAFs.

At both the start and end of the mixing period and following a 1-Hour standing period all loading rate WAFs were observed to have formed clear colorless media columns with test item floating at the media surface and dispersed throughout the media column.

Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present and hence it was considered appropriate to remove the WAFs by filtration through a glass wool plug and Postlip BW/S filter paper. Microscopic observations made after filtration showed there to be no dispersed test item present.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.

Total Organic Carbon Analysis

Analysis of the test preparations at 0 hours showed measured carbon concentrations to range from 3.41 to 5.38 mg C/L whilst concentrations in the range of 1.88 to 3.33 mg C/L were obtained at 72 hours. The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole the results were based on nominal loading rates only.

 

Table 5: Results of the TOC analysis

Samples	Nominal Loading Rate (mg/L)	Concentration of TOC (mg C/L)	Concentration of TOC Corrected for Control (mg C/L)
0 Hours	Control	<LOQ	-
	6.25	4.83	4.83
	12.5	3.42	3.42
	25	3.68	3.68
	50	3.41	3.41
	100	5.38	5.38
72 Hours	Control	<LOQ	-
	6.25	2.93	2.93
	12.5	1.88	1.88
	25	2.52	2.52
	50	2.15	2.15
	100	3.33	3.33

 

Validation of Mixing Period

Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic items, in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionized reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis. The results are summarized as follows:

Table 6:

Nominal Loading Rate (mg/L)	Time (hours)
	24 hours		96 hours
	mg C/L	mg C/L corrected for control	mg C/L	mg C/L corrected for control
Control	<LOQ	-	<LOQ	-
100	11.41	11.41	9.23	9.23

 

It is evident from this work that increasing the stirring period did not increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
This indicates that in the range of water solubility the test item was not toxic under the test conditions.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Exposure of Pseudokirchneriella subcapitata to the test item gave EL*50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. This indicates that in the range of water solubility the test item was not toxic under the test conditions. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. Given that a suitable method of chemical analysis could not be developed to measure the dissolved test item concentration present in the WAFs it was considered appropriate to take samples for Total Organic Carbon (TOC) analysis as an alternative. Analysis of the test preparations at 0 hours showed measured carbon concentration to range from 3.41 to 5.38 mg C/L whilst concentrations in the range of 1.88 to 3.33 mg C/L were obtained at 72 hours.

* EL = Effective Loading Rate

Description of key information

No effects up to the water solubility limit (OECD 201); read-across

Additional information

Since no studies investigating the toxicity of Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates (CAS 91052-16-3) to aquatic algae are available for this endpoint, in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5 a read across to the structurally related source substance Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6) was conducted. The target substance Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates (CAS 91052-16-3) and the source substance Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6) are considered to be structurally very similar. The difference between both substances is that the source substance is the potassium salt of the target substance.

Since both substances share common similar functional groups and neither the target nor the source substance consist of any structural alerts leading to a different toxicity profile this read-across is suitable. This read-across is further justified within the analogue justification in IUCLID Section 13. In this case of read-across, the best suited (highest degree of structural similarity, nearest physico-chemical properties) source substance was used for the assessment.

The study available for the read-across substance Glycerides, C16-18 mono-, di- and tri-, hydrogenated, citrates, potassium salts (CAS 91744-38-6) was performed according to OECD 201 (GLP) with the freshwater algae Pseudokirchneriella subcapitata under static conditions (Vryenhoef, 2012). Water accommodated fractions of nominal 6.25, 12.5, 25, 50 and 100 mg/L were prepared. No effects on growth rate of algae were recorded resulting in an EL50 (72 h) > 100 mg/L and a NOELR (72 h) ≥ 100 mg/L.

Based on the available result from one structurally related source substance (in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5) which is characterized by a comparable structure, it can be concluded that Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates will not exhibit acute toxicity to aquatic algae up to the limit of water solubility.