Short Chain Fructo Oligosaccharides

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:

20 Jun 2022 - 05 Aug 2022

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)

Version / remarks:

2006, Annex 5 corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Actilight 950P Premium "Fructo-Oligosaccharides"
Substance: Short-chain fructo-oligosaccharides
EC No.: 908-300-1
Appearance: White powder
Purity: 95.4%

COMPOSITION:
scFOS is short chains of fructose molecules linked to a molecule of sucrose (glucose-fructose disaccharide). Thus, scFOS is a multiconstituent substance composed of three oligosaccharides: 1-kestose (GF2), nystose (GF3) and fructosyl-nystose (GF4).
With:
- TOTAL FOS: 95.4 expressed as dry matter
- Kestose (GF2): 39.2% FOS
- Nystose (GF3): 47.6% FOS
- 1F-Fructofuranosylnystose (GF4:) 13.6% FOS
- Glucose +Fructose + Saccharose : 4.6% expressed as dry matter

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method: 2.0 mL from the approximate center of the test vessels at t=0 h and t=72 h
- Sample storage conditions before analysis: Samples were stored in a freezer (-20°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.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 22 mg/L but without algae (abiotic control) and samples for analysis were taken at the start and at the end of the test period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L. No other treatment than vigorous shaking was needed to completely dissolve the test material in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. 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.
- Control: Test medium without test item or other additives.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum at test initiation: 3 days
- Method of cultivation: 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.
- 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1 medium, according to NPR 6505, formulated using tap-water purified by reverse osmosis
- Pre-culture medium and test medium: M2 medium, according to OECD 201, formulated using tap-water purified by reverse osmosis.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

20 - 22°C

pH:

At t=0 h: 8.1
At t=72 h: 8.0 - 8.3

Nominal and measured concentrations:

Nominal concentrations: 4.6, 10, 22, 46 and 100 mg/L
Measured concentrations: 5.2, 10, 22, 46 and 106 mg/L at test start and remained stable, i.e. were at 85-104% relative to initial measured at the end of the test. Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations. (See 'Details on results' and Table 1 in 'Any other information on results incl. tables' for further details)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 10^4 cells/mL
- Control end cells density: 197 x 10^4 cells/mL
- Replicates: 3 replicates of each test concentration, 6 replicates of the control, 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Intervals of water quality measurement: pH: at the beginning and at the end of the test, for all test concentrations and the control. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 74 to 77 µE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the 100 mg/L test concentration and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Combined Limit/Range finding study test concentrations: 0.1, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, tested in a separate (non-GLP) study (performed March 2022)

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

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analytical results:
See Table 1 for details. No test material was detected in the samples taken from the control. The measured concentrations at the start of the test were 5.2, 10, 22, 46 and 106 mg/L for the nominal test concentrations 4.6, 10, 22, 46 and 100 mg/L respectively, i.e. 99-103% of nominal. During the exposure period, the concentrations remained stable, i.e. were at 84-109% of nominal and 85-104% relative to initial at the end of the test. Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations.

Biological results:
- Mean cell densities: See Table 2.
- Inhibition of growth rate: See Table 3. Inhibition of growth rates increased with increasing concentration of Fructo-oligosaccharides from 4.6 mg/L upwards resulting in 4.8% inhibition at 100 mg/L. Statistically significant inhibition of growth rates was found at test concentrations of 22 mg/L and higher.
- Inhibition of yield: See Table 4. Inhibition of yield increased with increasing concentration of Fructo-oligosaccharides from 4.6 mg/L upwards resulting in 22% inhibition at 100 mg/L. Statistically significant inhibition of yield was found at test concentrations of 22 mg/L and higher.
- Effect parameters: See Table 5.

Results with reference substance (positive control):

The batch of Raphidocelis subcapitata tested, showed expected sensitivity to Potassium dichromate (72-EC50: 0.95 mg/L with a 95% confidence interval ranging from 0.87 to 1.0 mg/L), based on the range specified in ISO International Standard 8692, February 2012 and based on the historical range of reference tests performed by the Test Facility in the last ten years.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Step-down Jonckheere-Terpstra Test, α=0.05, one-sided, smaller). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s Test) and a test for homogeneity of the variances (Levene’s test).
Calculation of ECx-values was based on a 3-parameter normal cumulative distribution function (CDF) using non-linear regression analysis with the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test material.
The EC50-value of the growth rate inhibition could not be determined because the observed effects were below 4.6%.
ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Analytical results

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]
	Nominal	Analyzed
0	0	<LOQ^a.	n.a.
	4.6	5.18	113
	10	10.3	103
	22	22.4	102
	22 ^b	23.0	104
	46	45.6	99
	100	106	106
72	0	<LOQ^a	n.a.
	4.6	4.52	98
	10	10.4	104
	22	23.3	106
	22 ^b	24.0	109
	46	38.6	84
	100	90.2	90

^a LOQ is defined as 0.10 mg/L

^b Without algae

 

Table 2: Individual and mean cell densities (x10^4 cells/mL)

Time	Replicate	Fructo-oligosaccharides Nominal conc. (mg/L)
		Control	4.6	10	22	46	100
0 h	1	1.000	1.000	1.000	1.000	1.000	1.000
	2	1.000	1.000	1.000	1.000	1.000	1.000
	3	1.000	1.000	1.000	1.000	1.000	1.000
	4	1.000
	5	1.000
	6	1.000
Mean:		1.000	1.000	1.000	1.000	1.000	1.000
Std.Dev.:		0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
24 h	1	9.292	6.933	7.386	6.856	7.794	7.852
	2	9.321	7.070	7.337	7.425	7.444	7.546
	3	9.540	7.702	7.634	6.909	7.011	7.249
	4	8.937
	5	9.433
	6	9.010
Mean:		9.255	7.235	7.452	7.063	7.416	7.549
Std.Dev.:		0.2366	0.4102	0.1592	0.3143	0.3922	0.3015
48 h	1	39.247	37.750	38.766	37.784	38.363	41.776
	2	41.508	39.656	41.751	41.265	40.074	40.988
	3	42.140	41.362	43.653	38.863	38.460	41.946
	4	41.226
	5	40.828
	6	40.964
Mean:		40.986	39.589	41.390	39.304	38.966	41.570
Std.Dev.:		0.9707	1.8069	2.4634	1.7819	0.9611	0.5111
72 h	1	178.466	184.981	133.350	159.119	174.387	150.747
	2	202.996	186.134	188.127	177.591	174.999	154.014
	3	201.352	185.035	198.945	173.983	184.228	154.014
	4	198.683
	5	198.435
	6	200.501
Mean:		196.739	185.383	173.474	170.231	177.871	152.925
Std.Dev.:		9.1120	0.6507	35.1669	9.7909	5.5135	1.8862

Table 3: Growth rate and percentage inhibition

Fructo-oligosaccharides Nominal conc. (mg/L)	Mean (day^-1)	Std. Dev.	n	%Inhibition
Control	1.760	0.0160	6
4.6	1.741	0.0012	3	1.1
10	1.714	0.0722	3	2.6
22	1.712	0.0195	3	2.7*
46	1.727	0.0102	3	1.9*
100	1.677	0.0041	3	4.8*

* Effect was statistically significant.

 

Table 4: Yield and percentage inhibition

Fructo-oligosaccharides Nominal conc. (mg/L)	Mean (x10^4 cells/mL)	Std. Dev.	n	%Inhibition
Control	195.739	9.1120	6
4.6	184.383	0.6507	3	5.8
10	172.474	35.1669	3	12
22	169.231	9.7909	3	14*
46	176.871	5.5135	3	9.6*
100	151.925	1.8862	3	22*

* Effect was statistically significant.

 

Table 5: Effect parameters

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	10	>100	>100	>100
Yield	Value	10	24	97	>100
	lower 95%-cl		11	61
	upper 95%-cl		49	>100

cl – confidence limit

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

The 72h-EC50 for growth rate inhibition (ERC50) and yield inhibition (EYC50) were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L. The 72h-NOEC for growth rate inhibition and yield inhibition equaled an analytically confirmed nominal concentration of 10 mg/L based on statistical significance.
The 72h-EC10 for growth rate inhibition (ErC10) and yield inhibition (EyC10) were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

Executive summary:

The toxicity of the ScFOS to the freshwater green algae Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201.

A full test was performed based on a preceding range-finding test. In this full test, 6 exponentially growing algal cultures were exposed to an untreated control, whereas 3 replicates per group were exposed to the nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L during 72 hours.

An analytical monitoring was carried out at t=0, t=72 hours for each test concentrations.

As a results, the concentrations of teach test solutions remained stable, i.e. were at 85-104% relative to initial at the end of the test. Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations.

Inhibition of growth rate and yield increased with increasing concentration of ScFOS from 4.6 mg/L upwards resulting in 4.8% growth rate inhibition and 22% yield inhibition at 100 mg/L. Statistically significant inhibition of growth rates was found at test concentrations of 22 mg/L and higher.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, ScFOS inhibited growth rate and yield of this freshwater algae species significantly at analytically confirmed nominal concentrations of 22 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ErC50) and yield inhibition (EyC50) were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

The 72h-NOEC for growth rate inhibition and yield inhibition equaled an analytically confirmed nominal concentration of 10 mg/L based on statistical significance.

The 72h-EC10 for growth rate inhibition (ErC10) and yield inhibition (EyC10) were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

Description of key information

The toxicity of the ScFOS to the freshwater green algae Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201.

Under the test conditions, ScFOS inhibited growth rate and yield of this freshwater algae species significantly at analytically confirmed nominal concentrations of 22 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ErC50) and yield inhibition (EyC50) were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

The 72h-NOEC for growth rate inhibition and yield inhibition equaled an analytically confirmed nominal concentration of 10 mg/L based on statistical significance.

The 72h-EC10 for growth rate inhibition (ErC10) and yield inhibition (EyC10) were beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

 

The toxicity of the test item to freshwater green algae Raphidocelis subcapitata was investigated in one GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.