Hexafluoropropene, oxidized, oligomers, reduced, fluorinated

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2014 to February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline-conform study conducted under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass and was not replaced.
At the end of the test, a sample was taken from the control and from the single test concentration to determine a potential impact of the test item on the algal cells. The shape and size of the algal cells were visually inspected.

For analytical verification, samples were analyzed immediately after sampling without any storage.

Vehicle:

no

Details on test solutions:

The test item is a complex mixture containing different sparingly soluble compounds. A dispersion of the test item was freshly prepared before the start of the exposure by direct addition of the test item to test water.
The test medium was prepared by directly adding 90 μL test substance to 1080 mL of test water (loading rate 140 mg/L) and using intense stirring by magnetic stirrers at room temperature in the dark over 4 hours (in closed system) to dissolve maximum amounts of the different compounds of the test item in the dispersion. Due to the volatile properties of the test item, the volume added to the test water was chosen to be great enough for practical handling; this resulted in a loading rate above 100 mg/L. Following the stirring period, emulsified substance was removed by centrifugation (3500 rpm for 15 minutes). The resulting supernatant was used as test medium. The test medium was prepared just before the start of the test.

GLP PRE-EXPERIMENTS
The solubility limit of the test item in test water and the appropriate procedure in order to get the maximum concentration achievable in the test media have been evaluated in two separate GLP pre-experiments.
Based on the results of both stirring experiments and in agreement with the Sponsor, a single centrifugation step and a stirring time of 4 hours were included in the study design of the definitive test.

1st Experiment
As the test item forms emulsions after stirring in water, the emulsified substance had to be removed by centrifugation to determine the water solubility of the test item in the test water.
To assess whether one centrifugation step would be sufficient or several centrifugation steps would be required, a pre-experiment was performed by stirring 50 μL of test substance in 110 mL of reconstituted test water according to ISO 6341 (see Section 3.3) in a closed system. The mixture was stirred for a period of approximately 7 hours. After the stirring period, the test item was allowed to settle (visible droplets on the bottom of the vessel) and sample aliquots were analyzed directly as well as after one or more centrifugation steps. Duplicate samples were taken, centrifuged at 3500 rpm for 10 minutes and an aliquot was analyzed for the content of GALDEN LMW. The centrifugation/analysis step was then repeated twice to determine whether the emulsified substance had been completely removed. The analytical results are reported in table 1.

The same stirring experiment was performed with AAP medium. The stirring period was approximately 7 hours and the samples were centrifuged once as this had been proven to be sufficient in the previous test with ISO medium.
The analytical results are reported in table 2.


2nd Experiment
In this stirring experiment, 65 μL of test substance were added to 580 mL of reconstituted test water (AAP medium) with a loading rate of 190 mg/L in a closed system. The approach included two different stirring periods of one and three hours. After the stirring periods, duplicate samples were taken from each replicate, centrifuged once at 3500 rpm for 10 minutes and analyzed for the content of GALDEN LMW. The analytical results are reported in table 3

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Age of inoculum (at test initiation): three days
- Method of cultivation: The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

After 24 hours until the end of the test, the analytically determined concentration of test item in the test medium was below the LOQ. The results were evaluated over an exposure period of 48h.

Post exposure observation period:

not applicable

Hardness:

The water hardness (calculated) of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

Test temperature:

24 °C

pH:

pH was 7.5
To keep the pH of the test media as constant as possible 6 mmol/L HEPES buffer (corresponding to 1430 mg/L) were added to the test water.

Salinity:

Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing.

Nominal and measured concentrations:

Nominal concentration = 140 mg/L.

Analytical concentration:
At the start of the test, the analytically determined concentration of GALDEN LMW in the test medium was 0.0939 mg/L. After 24 hours until the end of the test, the analytically determined concentration of GALDEN LMW in the test medium was below the limit of quantification (LOQ = 0.0357 mg/L).
(See analytical results and Table 4).
The biological results were related to the mean measured test item concentration calculated as the geometric mean of the test item concentrations measured at the start of the test, after 24 hours and after 48 hours (i.e. at the end of the test). When the measured concentration was below LOQ, the ½ LOQ (= 0.01785 mg/L) was used to calculate the mean measured concentration.

Details on test conditions:

TEST SYSTEM
- Type : closed system
- Test vessel: Glass flasks completely filled with test medium and tightly sealed with glass stoppers to avoid losses of test item by volatilization.
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 5000 cells/mL

- No. of vessels per concentration (replicates): six
- No. of vessels per control (replicates): six
- No. of vessels per vehicle control (replicates): not applicable (no vehicle used).

- Two additional flasks (satellite vessels) per each sampling time (24, 48 and 72 hours), one containing the test medium with algae and one without algae, were incubated under the test conditions (closed system) and only sampled for analytical verification. These separate flasks were only opened once for the analytical sampling at the defined sampling point, to keep the headspace as small as possible and, thus, minimize the loss of test substance through volatilization.

GROWTH MEDIUM
- Standard medium used: yes (AAP Medium)

TEST MEDIUM / WATER PARAMETERS
Reconstituted test water (AAP Medium) prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Light intensity and quality: The mean measured light intensity at the level of the test solutions was approximately 7800 Lux (range: 7200 to 8540 Lux, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15%-deviation from the average light intensity as recommended by the guideline.
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED :
- The algal biomass in the samples was determined by fluorescence measurement (BIOTEK Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, mission 680 nm). The measurements were performed daily for each flask. The measurements were performed at least in duplicate.

TEST CONCENTRATIONS
A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the algae up to its limit of solubility in the test water; the solubility limit in test water was determined in GLP - pre-experiments.

Reference substance (positive control):

not required

Remarks:

Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.031 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.031 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 140 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.031 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.031 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.031 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

At the start of the test, the analytically determined concentration of GALDEN LMW in the test medium was 0.0939 mg/L. After 24 hours until the end of the test, the analytically determined concentration of GALDEN LMW in the test medium was below the limit of quantification (LOQ = 0.0357 mg/L).
In accordance with the guideline, the results were evaluated over an exposure period of 48 hours to determine the NOEC, as the biomass increased by a factor of 58 during this time period and, therefore, the minimum multiplication factor of 16 was reached and the validity criterion fulfilled.

The biological results were related to the mean measured test item concentration calculated as the geometric mean of the test item concentrations measured at the start of the test, after 24 hours and after 48 hours (i.e. at the end of the test). When the measured concentration was below LOQ, the ½ LOQ (= 0.01785 mg/L) was used to calculate the mean measured concentration.

The test item had no statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 48 hours at the mean measured concentration of 0.031 mg/L with a loading rate of 140 mg/L (results of Student t-test, one-sided, α = 0.05). (Table 4 and Table 5)

The test item had a statistically significant effect on the growth rate μ and the yield Y of the algae after the test period of 72 hours (results of Student t-test, one-sided smaller, α = 0.05, Table 5 and Table 6), this observed effect was regarded as an artifact due to the inherent variability of the algae test system and not considered to be substance-related, as the measured concentration of the test item was already below LOQ after 24 hours of exposure. Therefore, the last test interval of 48 to 72 hours was not taken into account for the evaluation.

The mean measured test concentration of 0.031 mg/L (loading rate 140 mg/L) was, therefore, determined to be the 48-hour NOEC. This value might even be higher, but concentrations of the test item in excess of the solubility limit determined within the present study were not tested.
The 48-hour LOEC was greater than 0.031 mg/L, the maximum concentration of the test item that could be dissolved in the test water.
The microscopic examination of the algal cells after 72 hours showed no difference between the algae growing at the initially measured concentration of 0.0939 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item at this concentration.

Results with reference substance (positive control):

Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions.
The result of the latest positive control test performed in August 2014 (72-hour EC50 for the growth rate: 1.1 mg/L (Harlan Study Number D94966)) showed that the sensitivity of the test system was within the internal historical range of the 72-hour EC50 for the growth rate from 2000 to 2014: 0.71-1.7 mg/L.

Table 4. Analytical results

		Measured Concentration [mg/L]				Sample Preparation Factor	Determined Average Concentration of Test Item  [mg/L]
Sampling time	Sample description	Sample 1	Sample 2	Sample 3	Average
0 h	CONTROL loading rate: 0	*	*	*	na	1,6	< LOQ
	TEST vessel (+ algae) loading rate: 140 mg/L	0.0598	0.0606	0.0589	0.0598	1,6	0,0939
24 h	Satellite vessel + algae loading rate: 140 mg/L	*	*	0	na	1,6	< LOQ
	Satellite vessel - algae loading rate: 140 mg/L	*	*	0	na	1,6	< LOQ
48 h	Satellite vessel + algae loading rate: 140 mg/L	*	*	0	na	1,6	< LOQ
	Satellite vessel - algae loading rate: 140 mg/L	*	*	0	na	1,6	< LOQ
72h	Satellite vessel + algae loading rate: 140 mg/L	*	*	0	na	1,6	< LOQ
	Satellite vessel - algae loading rate: 140 mg/L	*	*	0	na	1,6	< LOQ
	CONTROL (+ algae) loading rate: 0	*	*	0	na	1,6	< LOQ

LOQ = 0.0357 mg/L

Table 5.

		Average growth rate μ and inhibion of μ (Ir)
		0 - 24h		0 - 48h		0 - 72h
Loading rate [mg/L]	Mean measured concentration (mg/L)	μ	Ir (%)	μ	Ir (%)	μ	Ir (%)
CONTROL	//	1,851	0	2,030	0	1,913	0
140 mg/L	0.031	1,915	-3,5	2,045	-0,7	1,834	4.1*

* mean value statistically significantly lower than in the control (according to Student t-test, one-sided smaller, α = 0.05), however, not considered as substance-related adverse effect, as the measured concentration of the test item was below LOQ already after 24 hours of exposure.

Table 6.

		Yield Y (x 10E3) and inhibition of Y (Iy)
		0 - 24h		0 - 48h		0 - 72h
Loading rate [mg/L]	Mean measured concentration (mg/L)	Y	Iy (%)	Y	Iy (%)	Y	Iy (%)
CONTROL	//	3,1	0,0	32,7	0,0	178,4	0,0
140 mg/L	0.031	3,3	-7,9	33,7	-3,1	140,7	21,1*

*mean value statistically significantly lower than in the control (according to Student t-test, one-sided smaller, α = 0.05), however, not considered as substance-related adverse effect, as the measured concentration of the test item was below LOQ already after 24 hours of exposure.

Validity criteria fulfilled:

yes

Conclusions:

The test item had no toxic effects on Pseudokirchneriella subcapitata up to its solubility limit in the test water.

Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006) and the Commission Regulation (EC) No 761/2009, C.3.

 

The test item is a complex mixture containing different sparingly soluble compounds. In order to assess its toxicity, a dispersion of the test item was freshly prepared before the start of the exposure by direct addition of the test item to test water. Since the test item was known to be volatile, the test was performed using glass flasks completely filled with test medium and tightly sealed with glass stoppers to avoid losses of test item (closed system).

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the algae up to its limit of solubility in the test water; the solubility limit in test water was determined in GLP - pre-experiments. Thus, a saturated solution of the test item was tested in the definitive test, together with a control group in parallel.

The test item was mixed into the test water (loading rate 140 mg/L) using intense stirring by magnetic stirrers at room temperature in the dark over 4 hours to dissolve maximum amounts of the different compounds of the test item in the dispersion. Following the stirring period, emulsified substance was removed by centrifugation (3500 rpm for 15 minutes). The resulting supernatant was used as test medium.

The preparation of the test medium was based on the OECD Guidance Document on Aquatic

Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

In order to define the test item concentration during the exposure period (72h) without affecting with losses for volatilization the concentration in the test vessels, two additional flasks (satellite vessels) at each sampling time (24, 48 and 72 hours) were incubated under the same test conditions as the test vessels (closed system) and only sampled once for analytical verification, in order to keep the headspace minimized.

Satellite vessels at each sampling time (24, 48 and 72 hours) include two replicates incubated with algae and two replicates incubated without algae.

 

At the start of the test, the analytically determined concentration of the test item in the test medium was 0.0939 mg/L. After 24 hours until the end of the test, the analytically determined concentrations of the test item in the test medium were below the limit of quantification (LOQ = 0.0357 mg/L).

In accordance with the guideline, the results were evaluated over an exposure period of 48 hours to determine the NOEC, as the algae biomass increased by a factor of 58 during this time period and, therefore, the minimum multiplication factor of 16 was reached and the validity criterion fulfilled.

The biological results were related to the mean measured test item concentration calculated as the geometric mean of the test item concentrations measured at the start of the test, after 24 hours and after 48 hours (i.e. at the end of the test). In addition, the results were reported based on the loading rate of the test item.

The 48-hour EC10, EC20, EC50values of GALDEN LMW could not be estimated because of theabsence of a significant inhibitory effect of the test item on the algal growth at the tested mean measured concentration of 0.031 mg/L (loading rate 140 mg/L).

 

The 48-hour NOEC was determined to be at the mean measured test concentration of 0.031 mg/L (loading rate 140 mg/L). This value might even be higher, but concentrations of the test item in excess of the solubility limit determined within the present study were not tested, in accordance with the test guidelines. The 48-hour LOEC was greater than 0.031 mg/L, the maximum concentration of the test item that could be dissolved in the test water.

In conclusion, the test item GALDEN LMW had no toxic effects onPseudokirchneriella subcapitataduring a 48-hour test period up to its solubility limit in the test water under thepresent test conditions, which was determined to be 0.031 mg/L with a loading rate of 140 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 October 2012 to 14 March 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

OECD Guideline-conform study conducted under GLP, however some limitations occurr due to technical difficulties in interpretation of results since the maximum concentration of GALDEN LMW achievable under the test conditions was lower than the water solubility value.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L (nominal)
- Sampling method: 50 ml of test item solutions and negative controls were collected from freshly prepared solutions at time 0 andd at the end of the test period (72 hours), after removing algal cells by centrifugation.

Triplicate samples of the test item solution and the negative control were taken at the indicated times from the test solutions, but only one sample was analysed, while the other were stored as retain samples to be analysed only if necessary.
No additional samplings after 24 and 48 hous were performed to avoid further test item loss during the sampling procedure and the mixing of the replicates. Also the analysis on a single replicate was not advisable, since enough homogeneity among the replicates could not be assured, being the system extremely instable.

Vehicle:

no

Details on test solutions:

An oversaturated test solution at 100 mg/L was prepared as follows:
Prior tot the test start, a 1-litre of flask of a 100 mg/L of Galden LMW solution was prepared by dosing with a micropipette 60 microliter of test item, corresponding to 100.2 mg of test item (considering its density: 1.65 -1.68 g/cm3, mean = 1.67 g/cm3), into 1002 mL of reconstituted water. The flask was tightly closed to prevent the test item evaporation.
The obtained solution appeared transparent with oily droplets on the bottom of the flask, therefore it was mixed for two hours by the mean of a magnetic stirred in dark conditions to achieve the maximum solubilisation of the test item. After stirring, it was settled down for two hours and then it was transferred into another flask, by siphoning it with a peristaltic pump, and then it was divided into the test flasks (and in vials for the analytical phase).
After pH check, test flasks were inoculated with algal culture..

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:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Source: cultured in the laboratories of the test facility and originally purchsed from the Insitute of Plant Physiology of the University of Gottingen, Germany.

The algae were cultured in a temperature controlled room at 24°C +- 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 weekly transferred to fresh medium and maintained in continuous shaking to ensure the necessary amount of CO2 and to keep the algae in suspension. Cultures containing deformed or abnormal cells were discarged. Only exponentially growing algal cultures have been used to start the test.

The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test in order to assure that the assy was performed with exponentially growing algal population. The cell density of the inoculum was around 10E6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Hardness:

not reported

Test temperature:

The room temperature under the light was continuously monitored during the course of the study by the mean of a data logger.
It was in the range 23.3 °C - 25.6 °C with a mean value of 24.4 °C and a standard deviation of 0.6 °C.
The maximum temperature was slight outside (0.1 °C) that the recommended range by OECD No. 201 for a veru short period (1 h).
This deviation was considered acceptable.

pH:

The pH values were measured with a portable pHmeter. For fresh solution, the pH values were measured sampling an aliquot immediately after test solution preparation, before pouring it in the flasks.
For spent solutions (after 72 h), pH were measured in each test flask.
At the beginning of the test, the pH value of the test medium was 7.75 for test item solution and 7.96 for the negative control. At the end of the test the pH value of the negative control ranged between 9.30-9.42 and the pH values of test item were in the range of 9.18 – 9.27.

The pH in the negative control varied of a maximum of 1.46 units, this value was considered acceptable since it was in accordance with the recommended limit by OECD No. 201 stating that trhe pH value in the negative control replicates should not increased more than 1.5 units during the test period.

Dissolved oxygen:

not measured

Salinity:

not applicable

Nominal and measured concentrations:

The test was carried out at the only nominal test item concentration of 100.0 mg/L, corrected for its purity (99.9% ). The 100.0 mg/L test solution was an oversaturated solution, in order to obtain the maximum solubility of Galden LMW under the test conditions.
The actual test concentration of the test item Galden LMW was analytically measured at the beginning and at the end of the test (72 hours).

The analytical recovery of the concentration of test item in the freshly prepared solution was 0.645 % of the nominal concentration of the oversaturated solution (100.0 mg/L), corresponding to 644.71 microgram/L.
After 72 hours of exposure, the analytical recovery was lower than the limit of detection of the analytical method. (Test item recovery are referred to the test item content, corrected for its purity, 99.9 %).
see table 1.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL capacity conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass
For the test concentration and for the negative control, 100 mL capacity conical glass flasks capped with air permeable stopper were used.

- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): n.a.
- Renewal rate of test solution (frequency/flow rate): n.a.

- Initial cells density: about 10E4 cells/mL
- Control end cells density: 418.0120 x 10E4 cells/mL (average measured value at 72 h)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n.a.

The test flasks were incubated in a temperature-controlled room, under continuous illumination and constant shaking. The flasks were randomly distributed under the light and were replaced every day during the test.


GROWTH MEDIUM
In deionised water (conducivity < 5 microScm-1) analytical grade salts were added to following final nominal concentrations:
NaHCO3: 50 mg/L
CaCl2 x 2H2O : 18.9 mg/L
NH4Cl : 15.0 mg/L
MgSO4 x 7H2O : 15.0 mg/L
MgCl2 x 6H2O : 12.0 mg/L
KH2PO4 : 1.6 mg/L

Trace Elements:
Na2EDTA x 2H2O : 100.0 microgram/L
FeCl3 x 6H2O : 64.0 microgram/L
MnCl2 x 4H2O: 415.0 microgram/L
H3BO3 : 185.0 microgram/L
Na2MoO4 x 2H2O : 7.0 microgram/L
ZnCl2 : 3.0 microgram/L
CoCl2 x 6H2O : 1.5 microgram/L
CuCl2 x 5H2O : 0.01 microgram/L


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not necessary
- Photoperiod: continuous illumination
- Light intensity and quality: 5893-6038 Lux
- Salinity (for marine algae): n.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter.
Algal cell density was measured every 24 hours taking aliquotes from each replicate of negative control and test item flasks, diluting them ina NaCl 9g/L solution and reading with an electronic particle counter.

TEST CONCENTRATIONS
- Test concentrations: 100 mg/L (nominal)
GALDEN LMW has a low water solubility. The nominal concentration of 100 mg/L was selected in order to obtain the maximum concentration of the test item.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: The reported value is the geometric mean deriving from the maximum concentrations of GALDEN LMW achievable under test conditions. No biological effects were observed.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: The reported value is the geometric mean deriving from the maximum concentrations of GALDEN LMW achievable under test conditions. No biological effects were observed.

Details on results:

No biological effects were observed at the maximun concentration of GALDEN LMW achiavable under test conditions.
See tables 2 and 3 for details on GROWTH RATE and YIELD.

Results with reference substance (positive control):

The algal sensitivity strain check was performed on July 2012 with 3,5-Dichlorophenol as reference substance.
- Results with reference substance valid? Yes
- EC50 (72h): 3.88 g/L; confidence interval 95%: 3.57 - 4.34 mg/L
- Reference data (UNI EN ISO 8692/2004) : 2.08 - 4.68 mg/L

Reported statistics and error estimates:

The CETIS v.1.026D software was used to carry out the statistical analysis: it automatically chooses the most appropriate analylisis for the pool of data.
The EyCx and the ErCx values were determined by a Linear Interpolation analysis.
The NOEC value for yield and growth rate endpoint were determined by comparison with the control, by Equal Variance t method.
As input data, the geometric mean of measured concentrations of test item Galden LMW was used.

Table 1. Analytical recovery of the measured test concentrations to the nominal ones.

Time (hours)	Nominal test item concentration (mg/L)*	Actual test item concentration (microgram/L)	Analytica Recovery ( % )*	Geometric mean (microgram/L)
0	100	644.71	0.645	4.40
72	100	0.03 **	//
*The analytical recoveries are referred to the test item content corrected for its purity, 99.9%.
** When tha measured concentration is lower than the L.O.D. of the analytical method, the L.O.D. itseld (= 0.03 microgram/L) is used for geometric mean calculation, as astated in OECD No.23 (2000)

Table 2. Algal growth rate and inhibiton caused by Galden LMW over 72 hours exposure in comparison with the negative control

Nominal concentration of Galden LMW	Mean Inhibition (%)
	0 - 24 Hours	0 - 48 Hours	0 - 72 Hours
0.0 (negative control)	//	//	//
100.0	2.7	- 3,2*	- 1,0*
* the algal growth rate was slightly higher than the engative control

Table 3. Algal yield inhibiton caused by Galden LMW over 72 hours exposure in comparison with the negative control

Nominal concentration of Galden LMW	Mean Growth inhibition (%)
0.0 (negative control)	//
100.0	- 6,0*
* The yield was slightly higher than in the negative control

Validity criteria fulfilled:

yes

Conclusions:

The test was conducted in order to obtain the maximum concentration of GALDEN LMW in water. No biological effects occurred at the maximum concentration of test item achievable under test conditions.

Executive summary:

The influence of the test item Galden LMW on the growth of the green algae speciesPseudokirchneriella subcapitata, formerly known asSelenastrum capricornutum, was investigated in a 72-hour limit test according to the OECD Guideline No. 201, 2011.

 

For this purpose, exponentially growing test algae were exposed for 72 hours to an aqueous test medium containing the test item at only nominal concentration of 100 mg/L, under defined conditions.

The 100.0 mg/L test solution was an oversaturated solution, in order to reach the maximum solubility of the test item.

Besides the concentration, a negative control without the test item was also prepared to check the acceptability of the algal inoculum.

The actual test concentration of the test item Galden LMW was analytically measured at the beginning and at the end of the test.

The analytical recovery of the concentration of test item in the freshly prepared solution was 0.645 % of the nominal concentration of the oversaturated solution (100.0 mg/L), corresponding to 644.71 microgram/L.

After 72 hours of exposure, the analytical recovery was lower than the limit of detection of the analytical method. (Test item recovery are referred to the test item content, corrected for its purity, 99.9 %).

The low concentrations obtained in algal growth medium were mainly due to:

- test item low solubility in test matrix;

- test item volatily.

The low % analytical recoveries (obtained compared to nominal test item concentration) were due to the preparation of an oversaturated solution, in order to reach the maximum test item concentration in algal growth medium, as suggested by OECD Guideline No. 23 (2000) on testing difficult substances.

Test solution preparation was strictly standardized in order to reduce test item evaporation.

 As reccomended by OECD guideline No. 23, the biological results were referred to the geometric mean of test item measured concentration, since the analytical recoveries of test item were lower than the range 80 % - 120 %.

 

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

 At the test end the cell density in the negative control increased on average by a factor of 418. This value complies with the validity criterion of the test , according to the mentioned guideline OECD No. 201, which indicates a minimum increased factor of 16.

The negative control also met the other validity criterion, with a coefficient of variation of daily growth rates at 72 hours of 11.4 % and a coefficient of variation of average growth in replicates negative control cultures during the test period of 1.2 %.

 

For the tested concentration, any significant biological effect was showed in comparison with negative control for both the endpoints, yield and growth rate, after 72 hours of exposure.

 The 72-hour EC10, EC20, Ec50 and NOEC, calculated in terms of geometric mean of measured concentrations and nominal test item concentration, for the two end-points, are reported in the following table:

ENDPOINT	0 - 72 h EC 10	0 - 72 h  EC 20	0 - 72 h  EC 50	0- 72 h  NOEC
Growth rate	> 4.40 microgram/L  > 100 mg/L *	> 4.40 microgram/L  > 100 mg/L *	> 4.40 microgram/L  > 100 mg/L *	4.40 microgram/L  100 mg/L *
Yield	> 4.40 microgram/L  > 100 mg/L *	> 4.40 microgram/L  > 100 mg/L *	> 4.40 microgram/L  > 100 mg/L *	4.40 microgram/L 100 mg/L *
* Nominal concentration

 

In conclusion, no biological effects were observed at the maximum concentrations of GALDEN LMW achievable under test conditions.

Description of key information

No biological effects occurred at the maximum concentration of GALDEN LW  achievable under test conditions.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.031 mg/L

Additional information

Two OECD Guideline-conform studies have been conducted in order to define the acute toxicity of GALDEN LMW to aquatic invertebrates. Both the studies have been performed under GLP, according to OECD guideline No. 201.

In the first test on Pseudokirchneriella subcapitata the concentration achieved under test condition was very lower than the nominal one (mean measured concentration = 0.004 mg/L, loading rate = 100 mg/L) and high decrease in concentration has been observed during the 72-h exposure.

These analytical results have been ascribed to the low water solubility of the substance and to losses for volatilization occurred during test solution handling and during the biological exposure.

Hence, a second study on Pseudokirchneriella subcapitata has been performed with a more refined study design (completely closed system) including a more refined test media preparation procedure defined basing on GLP pre-experiments, paying attention in avoiding any possible loss for volatilization during the test media handling.

In order to define the test item concentration during the exposure period (72h) without affecting the concentration in the test flasks with losses for volatilization, additional flasks (satellite vessels) at each sampling time (24, 48 and 72 hours) were incubated under the same test conditions as the test flasks and only used for analytical verification.

 

Again, during the biological exposure a high decrease in concentration has been observed during the 72-h exposure period and the concentration achieved under test condition was very lower than the nominal one (mean measured concentration = 0.031 mg/L, loading rate = 140 mg/L).

The losses over the test intervals have been ascribed to volatilization at sampling or to possible adsorption of the test item onto glass surfaces. Since the analytical verification in the test medium incubated without algae shows the same pattern of decrease in concentration, adsorption to biomass is expected to have, if any, a minor role in explaining the decrease in test item concentration over the test period.

 

In both the study, no biological effects have been observed at the limit of solubility of GALDEN LMW.