Hexafluoropropene, oxidized, oligomers, reduced, fluorinated

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the determination of the actual test item concentrations, four samples were taken from the test medium and from the control (one sample from each replicate) at the start and end of each test medium renewal period. All samples were analyzed immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
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 and prior to the test medium renewal by direct addition of the test item to test water.
The test medium was prepared by directly adding 20 μL test substance to 50 mL of test water for each of the four replicates (loading rate 676 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 10 minutes). The resulting supernatant was used as test medium. The test medium was prepared just before the introduction of the daphnids (i.e., at the start of the test and prior to the test medium renewal).

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.

2nd Experiment
In this stirring experiment, 65 μL of test substance were added to 580 mL of reconstituted test water according to ISO 6341 (see Section 3.3) 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 2.

Test organisms (species):

Daphnia magna

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

2.5 mmol/L
250 mg/L as CaCO3

Test temperature:

The test was performed in a temperature-controlled room. The water temperature was maintained at 20-21 °C with continuous monitoring.

pH:

pH of the test media and control were measured at the beginning and end of the test medium renewal periods.
The pH values of the test media were between 7.8 and 7.9.

Dissolved oxygen:

The test water was aerated prior to the start of the study until oxygen saturation was reached.
During the test period, the test water was not aerated. Dissolved oxygen concentrations in the test media and control were measured at the beginning and end of the test medium renewal periods.
The dissolved oxygen concentrations in the test media and control were at least 8.3 mg/L durig the test.

Salinity:

ISO 6341 reconsituted freshwater.

Nominal and measured concentrations:

At the start of the two test medium renewal periods, the analytically determined concentrations of GALDEN LMW in the test medium were 0.346 mg/L and 0.0961 mg/L, respectively.
At the end of two the test medium renewal periods, the analytically determined concentrations of GALDEN LMW in the test medium were 0.0738 mg/L and below the limit of quantification (LOQ), respectively. (analytical results are reported in Table 3).

Details on test conditions:

Since the test item was known to be volatile, the test was performed in 50-mL glass tubes completely filled with about 50 mL of test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance (closed system).
The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

TEST SYSTEM
- Test vessel: 50-mL glass tubes
- Type (delete if not applicable): closed (tightly sealed with glass stoppers to avoid losses of the volatile substance)
- Material, size, headspace, fill volume: 50 mL of test medium in every glass tube (no headspace)
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: The volume of test solution provided for each daphnid was 10 mL.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water according to ISO 6341
Ingredients:
CaCl2 × 2H2O: 294 mg/L
MgSO4 × 7H2O : 123 mg/L
NaHCO3: 65 mg/L
KCl : 5.8 mg/L
The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarities.

- Intervals of water quality measurement: at the beginnig and at the end of the renewal period (24h).

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Light intensity: between 800 and 1060 Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilization

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 test organisms up to its solubility limit.

Reference substance (positive control):

no

Remarks:

Potassium dichromate is tested as a positive control twice a year.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.102 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: *Arithmetic mean of the two geometric means determined of the test item concentrations measured at the start and end of each test medium renewal period. For measured concentrations < LOQ, ½ LOQ was used for calculation.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

676 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

> 0.102 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: *Arithmetic mean of the two geometric means determined of the test item concentrations measured at the start and end of each test medium renewal period. For measured concentrations < LOQ, ½ LOQ was used for calculation.

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

> 676 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

During the first 24 hours of the test, no immobilized test organisms were observed in the control and ain the test vessels.

After 48 hours of exposure, no immobilized test organisms were observed in the control and at the mean measured concentration of 0.102 mg/L (loading rate 676 mg/L). Therefore, the 48-hour EC0 and NOEC of the test item were both at the mean measured concentration of 0.102 mg/L (loading rate 676 mg/L). The 48-hour NOEC and the 48-hour EC0 might even be higher, but concentrations in excess of the solubility limit determined within the present study
were not tested. The EC50 and EC100 could not be estimated due to the absence of toxicity of the test item.

Results with reference substance (positive control):

Potassium dichromate is tested as a positive control twice a year.
The result of the latest positive control test in August 2014 showed that the sensitivity of the test organisms was within the historical range (EC50 (2000-2014) : 0.36-1.1 mg/L)

Table 3. Nominal concentrations

Sampling Day/ Sample Age	Loading Rate 676 mg Test Item/L	Measured Concentration					Sample Preparation Factor	Determined Average Concentration of Test Item
[day/h]		of  Test Item
		x
		Sample 1	Sample 2	Sample 3	Sample 4	Average
	[mg/L]	[mg/L]	[mg/L]	[mg/L]	[mg/L]	[mg/L]		[mg/L]
day 0 - 0 h	Control	*	*	*	*	n.a.	1.571.429	<LOQ
	test vessel	0.387	0.252	0.125	0.116	0.220	1.571.429	0.346
day 0 - 24 h	Control	*	*	*	*	n.a.	1.571.429	<LOQ
	test vessel	0.0837	0.0515	0.0142	0.0383	0.0469	1.571.429	0.0738
day 1 - 0 h	Control	*	*	*	*	n.a.	1.571.429	<LOQ
	test vessel	0.0560	0.0447	0.0439	0.100	0.0611	1.571.429	0.0961
day1 - 24 h	Control	*	*	*	*	n.a.	1.571.429	<LOQ
	test vessel	*	*	*	*	n.a.	1.571.429	<LOQ

In view of the low test concentrations as a result of the poor water solubility and the highly volatile nature of the test item, the losses over the test intervals may be due to adsorption of the test item onto glass surfaces and test organisms and due to volatilization at sampling. The differences in the test concentrations at the start of the two intervals may also be attributed to the inherent properties of the test substance

Validity criteria fulfilled:

yes

Conclusions:

The test item had no acute toxic effects on Daphnia magna up to its solubility limit in the test water.

Executive summary:

The acute toxicity of the test item toDaphnia magnawas determined in a 48-hour semi-static test according to the Commission Regulation (EC) No. 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

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 tubes 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 test organisms up to its limit of solubility in the test water.

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 water have been evaluated in two 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 676 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 10 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.

At the start of the two test medium renewal periods, the analytically determined concentrations of the test item in the test medium were 0.346 mg/L and 0.0961 mg/L, respectively. At the end of the two test medium renewal periods, the analytically determined concentrations of the test item in the test medium were 0.0738 mg/L and below the limit of quantification (LOQ = 0.0392 mg/L), respectively.

The losses over the test intervals could be due to adsorption of the test item onto glass surfaces and test organisms and due to volatilization at sampling.

The biological results were related to the mean measured test item concentrations calculated as the arithmetic mean of the two geometric means determined of the test item concentrations measured at the start and end of each test medium renewal period. In addition, the results were reported based on the loading rate of the test item.

After 48 hours of exposure, no immobilized test organisms were observed in the control and at the mean measured concentration of 0.102 mg/L (loading rate 676 mg/L). Therefore, the 48-hour EC0 and NOEC of the test item were both at the mean measured concentration of 0.102 mg/L (loading rate 676 mg/L). The 48-hour NOEC and the 48-hour EC0 might even be higher, but concentrations in excess of the solubility limit determined within the present study were not tested. The EC50 and EC100 could not be estimated due to the absence of toxicity of the test item.

In conclusion, the test item had no acute toxic effects on Daphnia magnaup to its solubility limit in the test water under the test conditions, which was determined to be 0.102 mg/L with a loading rate of 676 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Minor deviations, consireded not able to affect the results.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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 and at time 24 hours and from spent solutions at time 24 hours and at the end of the test.

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 glass beakers, hermetically closed (and in vials for the analytical phase).
pH and Oxygen concentration were checked and, since the values were within the provided range, the test solution was used for the test.

A negative control with reconstituted water only was also tested.

This procedure was repeated after 24 hours to renew the test solution and the negative control.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Daphnia magna Straus.
- Source: A strain was obtained from the supplier on December 2011, since that time the clone has been bred in the laboratories of the Test Facility.
- Age at study initiation (mean and range, SD): < 24 hours
- Feeding during test: no
- Food type during breeding: Organism were fed with a fixed amount of the laboratory cultured green algae Pseudokirchneriella subcapitata and with a suspension of the yeast Saccharomyces cerevisiae.


ACCLIMATION
Not necessary since the clone has been bred in the laboratories of the Test Facility under conditions identical to those of the test, with regards to temperature, light and water quality.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

According to the semi-static method the test solution was renewed after 24 hours from the beginning of the test.

Post exposure observation period:

no

Hardness:

140 -250 mg/L as CaCO3

Test temperature:

The incubation temperature was continuously monitored during the course of the study by the mean of a data logger. It was in the range 20.0 °C - 21.8 °C with a mean value of 21.2 °C and a standard deviation of 0.4 °C. The temperature range provided by OECD guideline is 20+- 2 °C, this range was corrected according to the datalogger precision (therefore the valid range to be considered for the test was 18.5 – 21.5 °C ) while temperature values were corrected according to correction factor.
The temperature variation during the test period was higher than the maximum recommended variation of 1.0 °C (1.8 °C) and temperature values slightly exceeded the recommended ; this deviation was however considered acceptable because it happened for short periods during the course of the test and no mortality was shown.

pH:

The pH of the test medium after preparation was measured with a pHmeter. pH values were 7.42 – 7.55 for test item solutions and 7.35 – 7.25 for negative control. The pH did not vary by more than 0.34 unit during the test period, as required in the mentioned guidelines.
For fresh solutions, pH was measured sampling an aliquote immediately after test solution preparation, before pouring it in the test glass beakers.
For spent solutions, pH was measured after removing the daphnids from the test glass beakers containing spent solutions.

Dissolved oxygen:

The dissolved oxygen concentration the test medium and in the negative control was measured with a portable oxymeter and it was always higher than 6.0 mg/L.
For fresh solutions, oxygen concentration was measured sampling an aliquote immediately after test solution preparation, before pouring it in the test glass beakers.
For spent solutions, oxygen concentration was measured after removing the daphnids from the test glass beakers containing spent solutions.

Salinity:

The following salts were dissolved in purified water in the following nominal concentrations:
CaCl2 x 2H2O = 294 mg/L
MgSO4 x 7H2O = 123 mg/L
NaHCO3 = 65 mg/L
KCl = 5.8 mg/L

Nominal and measured concentrations:

The main 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 analytical concentration of Galden LMW was measured at time 0 and 24 hours in the freshly prepared solutions, and at time 24 hours and at the end of the test in the spent solutions.
See table 1 for details on Recoveries.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type (delete if not applicable): closed
- Renewal rate of test solution (frequency/flow rate): after 24 hours from the beginning of the test
The test was performed in 100 mL glass beakers filled with 40 mL of test medium. The beakers were hermetically closed to reduce test item evaporation, water loss and to avoid dust entry into the solutions.
- Aeration: Prior to study start, the test water was areated until oxygen saturation. During the test period, the test water was not areated.

- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): n.a.
The 20 daphnids were randomly distibuited to the test vessels at test initiation and transferred in the fresh prepared solutions after 24hours.
The daphnid's loading rate was lower than one daphnid for 2 mL of solution, as required by OECD guideline No.202

TEST MEDIUM / WATER PARAMETERS
For reconstituted test water preparation, analytical grade salts were dissolved in purified water until obtaining the following nominal concentration:
CaCO2 x 2 H2O : 2.0 mmol/L (= 294 mg/L)
MgSO4 x 7H2O : 0.5 mmol/L ( = 123 mg/L)
NaHCO3 : 0.075 mmol/L ( = 65 mg/L)
KCl : 0.075 mmol/L ( = 5.8 mg/L)
- Ca/Mg ratio: 4:1 (based on molarity)
- Na/K ratio: 10:1 (based on molarity)

The pH values and dissolved oxygen concentrations in the test media and in the negative controls were daily measured in fresh and spent solutions.

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary
- Photoperiod: 16 hours light / 8 hours darkness with 30-min transition period
- Light intensity: 892 -924 Lux (measured values)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The Daphnids were observed for immobility after 24 and 48 hours of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile). The immobilization percentage was calculated.


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:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

43.19 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

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:

48 h

Dose descriptor:

IC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

IC50

Effect conc.:

> 43.19 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

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

Details on results:

No biological effects were observed at the maximun concentration of GALDEN LMW achiavable under test conditions.

Results with reference substance (positive control):

Daphnia clone sensitivity check was performed on July 2012 with Potassium dichromate as reference substance.
- Results with reference substance valid? YES
- IC50 (24h) : 0.6 mg/L; confidence interval 95%: 0.6 - 0.6
- Reference data (UNI EN ISO 6341/99) : 0.6 - 2.1 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 analysis for the pool of data. The IC0 and IC100 values were determined directly from Raw Data at each observation time.
The IC 50 value at 24 and 48 hours was determined by statistical analysis with Linear Interpolation analysis.
The NOEC value at 24 and 48 hours was determined by comparison with the control by Fisher's Exact method.
As input data, the measured geometric mean of test item Galden LMW was used.

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

Time (hours)	Nominal test item concentration (mg/L)*	Actual test item concentration (microgram/L)	Analytica Recovery ( % )*
0	100	184.51	0.185
24 fresh	100	29.55	0.030
24 spent	100	115.06	0.115
48	100	5.54	0.006
*The analytical recoveries are referred to the test item content corrected for its purity, 99.9%.

Table 2.Results in terms of measured geometric mean concentration and of nominal test item concentration

Time intervals (hours)	IC 0	IC 100	IC 50	NOEC
0 - 24	73.84 microgram/L 100 mg/L *	> 73.84 microgram/L 100 mg/L *	> 73.84 microgram/L 100 mg/L *	73.84 microgram/L 100 mg/L *
0 - 48	43.19 microgram/L 100 mg/L *	> 43.19 microgram/L 100 mg/L *	> 43.19 microgram/L 100 mg/L *	43.19 microgram/L 100 mg/L *
* Nominal concentration

Validity criteria fulfilled:

yes

Conclusions:

The test was conducted in order to obtain the maximum concentration of GALDEN LMW in water. Due to the substance properties (low water solubility and high volatility) the analytical concentration in water was very lower than the nominal one (100 mg/L). No biological effects occurred at the maximum concentration of test item achievable under test conditions.

Executive summary:

The acute toxicity of the test item Galden LMW to Daphnia magna was determined in a 48-hour semi-static test according to the OECD Guideline No. 202 (2004), to UNI EN ISO 6341:2004 and to Council Regulation EC 440/2008 (C.2.).

 

For this purpose, 20 juvenile daphnids (< 24 hours old at the initiation of the test) were exposed to an aqueous test medium containing the test item at the only nominal concentration of 100.0 mg/L.

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

 

The exposed organism, were checked for immobilization 24 and 48 hours after test initiation.

According to the semi-static method the test solution was renewed after 24 hours from the beginning of the test.

 

The actual test concentration of the test item Galden LMW was analytically measured at the beginning of the test, after 24 hours (in the freshly prepared and in the spent solutions) and at the end of the test period.

 

In fresh solution the analytical recoveries of the concentration of the test item were respectively 0.185 % and 0.115 % of the nominal concentration of the oversaturated solution (100.0 mg/L), corresponding to 184 microgram/L and 115.06 microgram/L respectively.

In the spent test item solution (24 and 48 hours after the beginning of the test) the test item concentration decreased; the analytical recoveries were respectively 0.030 % and 0.006 % of the nominal values, corresponding to 29.55 microgram/L and 5.54 microgram/L respectively. (The analytical recoveries are referred to the test item content corrected for its purity, 99.9% )

The low concentration obtained in test solutions were mainly due to:

- test item low solubility in the test item;

- test item high volatility.

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 reconstituted water, as suggested by OECD No. 23 (2000) on testing difficult substances.

Test solutions preparation was strictly standardized in order to get the maximum reproducibility and to reduce the test item evaporation. The toxicity test on Daphnia magna was conducted in hermetically closed containers.

As recommended by OECD Guidance Document on testing difficult substances No. 23 (2000), the biological results were referred to the geometric mean of test item measured concentrations, since the analytical recoveries of test item were lower than the range 80 - 120 %.

At the end of test period, in the negative control 0 % of immobilization was observed and no daphnid showed signs of disease or stress (e.g. to be trapped on the test water surface). These values comply with the validity criteria of the test, that required a maximum immobilization or a maximum number of daphnids showing disease or stress signs of 10 % in the negative control medium at the end of the test.

The obtained experimental results showed that no biological effect occurred at the maximum concentration of test item which could be reached in reconstituted water.

IC0, IC100, IC50 values and the NOEC at 24 and 48 hours were calculated and the results, in terms of geometric mean of measured concentration and nominal test item concentration, were as follow:

Time (hours)	IC 0	IC 100	IC 50	NOEC
24	73.84 microgram/L 100 mg/L *	> 73.84 microgram/L 100 mg/L *	> 73.84 microgram/L 100 mg/L *	73.84 microgram/L 100 mg/L *
48	43.19 microgram/L 100 mg/L *	> 43.19 microgram/L 100 mg/L *	> 43.19 microgram/L 100 mg/L *	43.19 microgram/L 100 mg/L *
* Nominal concentration

 

The IC50 and NOEC values were determined from the Raw Data by the statistical analysis in comparison with the negative control, while IC0 and IC100 at each observation time were directly extracted from Raw Data.

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 LWM  achievable under test conditions.

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. 202.

In the first test (48h, semi-static) the concentration achieved under test condition was very lower than the nominal one (mean measured concentration = 0.043 mg/L, loading rate = 100 mg/L) and high decrease in concentration has been observed during the 24-h renewal periods.

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 exposure.

Hence, a second study (48h, semi-static) 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.

Again, during the biological exposure a high decrease in concentration has been observed during the 24-h renewal periods and the concentration achieved under test condition was very lower than the nominal one (mean measured concentration = 0.102 mg/L, loading rate = 676 mg/L).

The losses over the test intervals have been ascribed mainly to volatilization at sampling and to possible adsorption of the test item onto glass surfaces. At the moment there is no evidence about the possible adsorption on the test organisms, considering the low biomass/water rate in the test vessels, if adsorption on Daphnia occurred this is expected to have had a minimum effect on the decrease in concentration.

 

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