Sodium olivoyl glutamate

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:

2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment No 23 - Guidance Document On Aquatic Toxicity Testing Of Difficult Substances And Mixtures ENV/JM/MONO(2019).

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SODIUM OLIVOYL GLUTAMATE
Common name: Sodium olivoyl glutamate
Chemical name: Glutamic acid, N-olivoyl derivs., sodium salts
CAS number: 1628321-85-6
EC number: 944-266-4

SOURCE OF TEST MATERIAL
Supplier: Sponsor
Batch number: 2072900-M
Expiry date: 29/07/2023
Purity: nominal content 100.0%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage condition: at room temperature, light protection not needed.

Analytical monitoring:

yes

Details on sampling:

The concentration of SODIUM OLIVOYL GLUTAMATE in the test medium was analytically measured according to a fully validated analytical method (study CH - 0926-2022). Samples of test media were taken, in duplicate, at time 0 and after 72 hours. Additional replicates used only for sampling (and analysis) at 24 and 48 hours were performed to follow the decrease of test item concentration during test period. Final samples were obtained by mixing the replicate test vessels media together. Control samples were analysed at the same time to verify the absence of test item contamination.

Vehicle:

no

Details on test solutions:

Prior to the test start, a 100.0 mg test item/L stock solution (SS1) was prepared by direct weighting 0.0500 g of test item into 500 mL of algal medium. The stock solution was clear and transparent with slightly foam on the flask’s meniscus. Starting from stock solutions (SS1), the other water test solutions were prepared transferring proper volumes of SS1 in algal medium. Test solutions was clear and transparent. A negative control (with algal growth medium only) was also tested.

Test organisms (species):

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

Details on test organisms:

The study was performed with the unicellular, fresh-water green algae Raphidocelis subcapitata strain SAG 61.81 (also known as Pseudokirchneriella subcapitata), cultured in the laboratories of the Test Facility and originally purchased from the Institute of Plant Physiology of the University of Göttingen, Germany. The algae were cultured in a climatic chamber at 24 ± 2°C under continuous uniform illumination in the range 4440 - 8880 Lux in the spectral range 400-700 nm. The culture medium was the same of the test medium; the stock cultures were transferred weekly to fresh medium and maintained in continuous shaking to ensure the necessary amount of CO2 and to keep algae in suspension. Cultures containing deformed or abnormal cells were discarded. Only exponentially growing algal cultures were used to start the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Deionised water was used, with the addition of salts as described under "Salinity".

Test temperature:

The incubation temperature was continuously monitored during the study by a probe installed in the environmental test chamber. It was in the range 23.89 – 24.20°C with a mean value of 23.99°C and a standard deviation of 0.04°C.
In agreement with the temperature range provided by OECD guideline 202, 2004 (24 ± 2°C), the temperature values during the test were within the provided range and did not vary more than 1°C during the test period.

pH:

Test medium pH was measured with a portable pH meter at the beginning and end of the test. For fresh solutions, the pH values were measured in an aliquot sampled immediately after test solutions preparation, before pouring it in the test flasks. For spent solutions (after 72 hours), pH was measured in each test flask. The pH measured during the test period are reported below.

Nominal test item concentration (µg/L) Start (0 hours) pH End (72 hours) pH (mean values)
0.00 (negative control) 7.95 7.67
4.3 7.91 7.61
9.4 7.88 7.66
20.7 7.88 7.69
45.5 7.89 7.81
100.0 7.84 7.85

Dissolved oxygen:

Not determined.

Salinity:

The salinity of test medium is defined by the parameters used to obtain the reconstituted water. In deionised water (conductivity < 5 μScm-1) analytical grade salts will be added to following final nominal concentrations:

Macro-nutrients:
NaHCO3 50.0 mg/L
CaCl2 x 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 x 7 H2O 15.0 mg/L
MgCl2 x 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L

Trace Elements:
Na2EDTA x 2 H2O 100.0 μg/L
FeCl3 x 6 H2O 64.0 μg/L
MnCl2 x 4 H2O 415.0 μg/L
H3BO3 185.0 μg/L
Na2MoO4 x 2 H2O 7.0 μg/L
ZnCl2 3.0 μg/L
CoCl2 x 6 H2O 1.5 μg/L
CuCl2 x 2 H2O 0.01 μg/L

pH 8.1 ± 0.2

Conductivity:

Conductivity value of used deionised water was < 5 μScm-1. Test medium conductivity was not measured.

Nominal and measured concentrations:

Analytical recovery of test item measured concentrations

Nominal test item* concentrations (mg/L)
Dose level 1 - at 0, 24, 48, 72 hours: 4.3
Dose level 2 - at 0, 24, 48, 72 hours: 9.4
Dose level 3 - at 0, 24, 48, 72 hours: 20.7
Dose level 4 - at 0, 24, 48, 72 hours: 45.5
Dose level 5 - at 0, 24, 48, 72 hours: 100

Actual test item concentration (mg/L)

Dose level 1 - at 0, 24, 48, 72 hours: 4.51 - 2.12 - Dose level 2 - at 0, 24, 48, 72 hours: 8.48 - 4.74 - Dose level 3 - at 0, 24, 48, 72 hours: 17.44 - 12.59 - 1.32 - n.d.
Dose level 4 - at 0, 24, 48, 72 hours: 40.30 - 30.52 - 1.65 - Dose level 5 - at 0, 24, 48, 72 hours: 91.20 - 92.35 - 6.82 -
Analytical recovery (%)

Dose level 1 - at 0, 24, 48, 72 hours: 104.9 - 49.4 - / - /
Dose level 2 - at 0, 24, 48, 72 hours: 90.2 - 50.4 - / - /
Dose level 3 - at 0, 24, 48, 72 hours: 84.2 - 60.8 - 6.4 - /
Dose level 4 - at 0, 24, 48, 72 hours: 88.6 - 67.1 - 3.6 - /
Dose level 5 - at 0, 24, 48, 72 hours: 91.2 - 92.3 - 6.8 - /

Geometric means of test item measured concentrations

Test item concentration (mg/L) - 4.3 - 9.4 - 20.7 - 45.5 - 100.0
Geometric mean (mg/L) T0-T24 h - 3.10 - 6.34 - 14.82 - 35.07 - 91.77
Geometric mean (mg/L) T0-T48 h - 1.13 - 1.82 - 6.62 - 12.67 - 38.59
Geometric mean (mg/L) T0-T72 h - 0.62 - 0.88 - 2.32 - 4.18 - 9.64


*according to 100.0 % purity.
LOQ: Limit of quantification (0.29 mg/L)
n.d.: not detected, lower than LOD (0.10 mg/L)

Details on test conditions:

TEST SYSTEM
All the described procedures were conducted in a laminar flow cabinet to ensure the sterility of the algal cultures. For the test item concentrations and the negative control, 100 mL capacity conical glass flasks capped with air permeable stoppers were used. Three replicates for test concentrations and six for negative were prepared. At test start, 50 mL of the relevant test solution was poured into each test flask and inoculated with algae to obtain a starting cell density of about 104 cells/mL. The inoculum was taken from a pre-culture started 3 days before the test beginning (containing approximately 106 cells/mL), in order to ensure that the assay was performed with an exponentially growing population.

OTHER TEST CONDITIONS
The test flasks were incubated in a controlled environment chamber, under continuous illumination and constant shaking. The flasks were randomly distributed under the light and moved every day during the test. Light intensity ranged between 6987 and 7505 Lux.

EFFECT PARAMETERS MEASURED
Algal cell density was measured every 24 hours by taking aliquots from each test concentration replicate and the negative control, diluting them in a NaCl 9 g/L solution and reading with an electronic particle counter.

RANGE-FINDING STUDY
The test concentrations were chosen based on the results of a preliminary toxicity screening (no GLP preliminary test), in which the effects were studied at 1, 10 and 100 mg/L (test item nominal concentrations) and effects were observed at the higest dose level.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol as reference item

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 45.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 45.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 4.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 4.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 20.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 20.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 2.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 2.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.88 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 20.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 5.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 2.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 33.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 13.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 3.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae (as may be caused by the exposure to the test substance) at the end of the test. No remarkable observations were made concerning the appearance of the cells of the inoculum culture and of the test item treated algae.
Since the analytical recoveries were not in the range 80% - 120% of the nominal concentrations during the test period, the biological results were referred also as geometric mean of test item
measured concentrations, as recommended by OECD Guideline No. 201.

Results with reference substance (positive control):

The reference substance showed an ErC50 at 72 hours of 3.63 mg/L (CI 95% 3.43-3.81) in the internal laboratory test performed on the same alga strain a few months before the current study. This result is in full agreement with published data and demonstrates that the used alga strain has an adequate level of sensitivity to that substance.

Reported statistics and error estimates:

NOEC and LOEC values were calculated by Bonferroni Adj t Test, after verifying data variances and data distribution by Variance ratio F and by Shapiro Wilk W Normality test. In all cases equal variance and normal distribution were observed.

 

 Effect of test item on the growth of Raphidocelis subcapitata (also known as Pseudokirchneriella subcapitata)

Nominal test item concentrations (mg/L)	Replicate	Cell density (n. cells/mL) (*)
		24 h	48 h	72 h
0.0 (negative control)	A	55287	282800	1849067
	B	56117	279100	1819267
	C	64287	373200	2709267
	D	62257	335900	2752067
	E	61907	325400	2949667
	F	61077	315500	2237867
	Average value	60155	318650	2386200
4.3	A	83127	388600	2815467
	B	74447	508600	3344667
	C	70127	393400	3206067
	Average value	75900	430200	3122067
9.4	A	76427	434700	2543067
	B	75897	466000	3156867
	C	76517	465100	3567667
	Average value	76280	455267	3089200
20.7	A	77047	475300	2400867
	B	73877	422300	1937867
	C	76177	449300	2506867
	Average value	75700	448967	2281867
45.5	A	71547	336100	1214867
	B	68637	356300	1504667
	C	75747	302000	1198467
	Average value	71977	331467	1306000
100.0	A	66787	165340	309333
	B	67447	170140	254533
	C	69587	174100	326133
	Average value	67940	169860	296667

(*) Cell density at time 0 h: 10⁴ cell/mL.

 

Growth rate

The calculated growth rates and the percentage of growth inhibition for the tested concentrations are reported in the following table.

 Algal growth rate and percentage inhibition

Nominal test item concentration (mg/L)	0-24 h		0-48 h		0-72 h
	Mean growth rate	Mean inhibition (%)	Mean growth rate	Mean inhibition (%)	Mean growth rate	Mean inhibition (%)
0.0 (negative control)	1.7928	-	1.7282	-	1.8189	-
4.3	2.0243	-12.9	1.8769	-8.6	1.9137	-5.2
9.4	2.0318	-13.3	1.9089	-10.5	1.9078	-4.9
20.7	2.0240	-12.9	1.9016	-10.0	1.8080	0.6
45.5	1.9729	-10.0	1.7493	-1.2	1.6222	10.8
100.0	1.9159	-6.9	1.4161	18.1	1.1282	38.0

 

E_rC₁₀, E_rC_20, E_rC₅₀ values, NOEC and LOEC expressed in terms of nominal test item concentrations

Time (h)	ErC10 (mg/L)	ErC20 (mg/L)	ErC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
0 – 24	> 100.0	> 100.0	> 100.0	100.0	> 100.0
0 – 48	55.1 (44.5 – 62.5)	85.4 (75.9 – 93.0)	> 100.0	45.5	100.0
0 – 72	33.9 (24.3 – 49.3)	55.0 (49.0 – 62.5)	> 100.0	20.7	45.5

 

The results in terms of geometric means of test item measured concentrations

Time (h)	ErC10 (mg/L)	ErC20 (mg/L)	ErC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
0 - 24	> 91.77	> 91.77	> 91.77	91.77	> 91.77
0 - 48	16.69 (12.43 – 20.44)	30.97 (26.31 – 35.2)	> 38.59	12.67	38.59
0 - 72	3.38 (2.60 – 4.17)	5.15 (4.50 – 5.71)	> 9.64	2.32	4.18

 

Yield

The calculated yield and the percentage of yield inhibition for the tested concentrations are reported in the following table.

Algal yield and percentage inhibition

Nominal test item concentration (mg/L)	Mean yield at 72h (cell/mL)	Mean yield inhibition (%)
0.0 (negative control)	2376200	-
4.3	3112067	-31.0
9.4	3079200	-29.6
20.7	2271867	4.4
45.5	1296000	45.5
100.0	286667	87.9

 

 E_yC₁₀, E_yC_20, E_yC₅₀ values, NOEC and LOEC expressed in terms of nominal test item concentrations

Time (h)	EyC10 (mg/L)	EyC20 (mg/L)	EyC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
0 – 72*	13.9 (10.0 – 25.8)	20.4 (10.7 – 29.5)	41.0 (30.1 – 54.9)	20.7	45.5

 

The results in terms of geometric means of test item measured concentrations

Time	EyC10 (mg/L)	EyC20 (mg/L)	EyC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
72 h	1.48 (0.77 – 3.32)	2.28 (1.00 – 3.22)	3.88 (3.14 – 4.85)	2.32	4.18

Validity criteria fulfilled:

yes

Remarks:

All validity criteria (increase of cell density, coefficient of variation of daily section-by-section growth rates and coefficient of variation of average growth) were met.

Conclusions:

The test perfomed showed the following results:
- the 72 hr ErC 50 (for algae) is > 9.64 mg/L (Growth rate)
- the chronic NOEC (for algae) is 2.32 mg/L (Growth rate).

Executive summary:

A full dose-response test was performed to evaluate the effect of the test item on Raphidocelis subcapitata (also known as Pseudokirchneriella subcapitata).

Since the analytical recoveries were not in the range 80% - 120% of the nominal concentrations during the test period, the biological results were referred as geometric mean of test item measured concentrations, as recommended by OECD Guideline No. 201

The test perfomed showed the following results:
- the 72 hr ErC 50 (for algae) is > 9.64 mg/L (Growth rate)
- the chronic NOEC (for algae) is 2.32 mg/L (Growth rate).

Description of key information

The test perfomed showed the following results:
- the 72 hr ErC 50 (for algae) is > 9.64 mg/L (Growth rate)
- the chronic NOEC (for algae) is 2.32 mg/L (Growth rate).