Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 - 26 January 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guidleline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Protocols according to the ecotoxicological testing of petroleum products by CONCAWE 1992 and for aquatic toxicity testing of lubricants by ASTM (standard protocol 06081-97). These comply with the general principles of OECD guidelines.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Trade Name: Zonyl® PFBE

Chemical Name: 3;3,4,4,5,5,6,6,6-nonafluorohexene

CAS Number: 19430-93-4

Lot Number: 335

Purity: 96%

Water solubility: negligible

Vapour pressure: 0.2 bar (20 °C)

Chemical stability (water/light): stable

Specific density (20°C): 1.418 kg/L

Physical state and appearance: colourless, liquid

Boiling point: 58°C

Saftey data sheet: yes

Waste disposal: aqueous organic waste

Storage conditions: 4°C in closed containers
Prevent spills and avoid open flames and keep away from heated surfaces above 200° C.

Expiry date: 2 years

Further information: none

Test material preparation and addition: WAF preparation

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The concentration of the test substance in the WAFs was analysed at the beginning and the end of the exposure (72 h) of the algae.

100 ml samples of each WAF were taken for the analytical measurements. At the end of the toxicity test periods, the WAF test media were filtered through a plastic sieve to remove any particles. The sieve was rinsed with 10 ml of the corresponding WAF before filtration.

Test solutions

Details on test solutions:
The WAF preparation was performed as described in the CONCAWE test protocol and in the ASTM standard 06081-97, with the modifications given below. For each test concentration, a WAF was prepared.

The water-accommodated fractions (WAFs) were prepared by mixing the test substance with the dilution water at loading rates of 100 mg/L and 1000 mg/L in clean cylindrical mixing vessels. In contrast to the CONCAWE test protocol and to the ASTM standard 06081-97, the mixing vessels were not equipped with a drain port near the bottom for drawing off the WAF as the density of the test substance was above 1 gl/ml. So the aqueous WAF phase was drawn off after WAF preparation through the upper orifice.
The vessels were filled to a maximum without headspace. The containers were sealed with teflon covered screw caps. The vessels were tightly sealed to prevent loss of volatiles. The vessels were protected from light with tinfoil to prevent photochemical degradation of dissolved components.

A magnetic stirring bar was placed in each vessel. The test substance was added to the bottom of the vessels being careful not to contaminate the orifice and the side walls. Then the appropriate volume of water was added. Mixing was initiated with the vortex in the centre, extending maximally 1/3 rd (10 - 35 % of vessel depth) from the top to the bottom of the vessel. It was as low as possible to maintain mixing of the water phase. Observations of the vortex-depth and mixture appearance were documented.
A mixing period of 24 hours was found to be sufficient for equilibration.
Following mixing, the contents of the vessels were allowed to stand undisturbed for 1 hour to allow separation of the aqueous and undissolved phases. The aqueous phase (the WAF) was then taken out of the orifice and filled into the test vessels for toxicity testing. The first portion of WAF was used to rinse the vessels in order to saturate the surfaces. After filling, the vessels were sealed immediately and only opened again to introduce the test organisms and again at the end of the test. Another portion was filled in separate vessel for chemical analysis.
Care was taken to ensure that any undissolved material was not transferred to the test vessels. The WAFs were not stored for more than 1 - 2 hours prior to testing. Storage was always in completely full sealed vessels, in the dark at 4°C if stored for periods longer than 2 hours.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism

Specification: Selenastrum capricornutum, Chlorophycea, Chlorophyta.
Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches lnstitut of the University at Goittingen, Albrecht von Haller lnstitut, Untere
Klarspule 2, D-37073 Gottingen. Catalog No 61.81.
Cultivation: The stock cultures were maintained fulfilling the criteria of the OECD Test Guideline 201. Prior to testing, a pre-culture was established in test medium to obtain exponentially-growing algae for the test .

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
enhance limit test

Test conditions

Test temperature:
22 ± 1°C
pH:
Control: 9.09 - 9.30
100 mg/l loading rate: 8.74 - 9.04
1000 mg/l: loading rate: 8.74 - 9.14
Nominal and measured concentrations:
Control: 0 mg/l (loading rate); <0.05 mg/l (measured conc)
100 mg/l (loading rate); 0.92 mg/l (arithmetic mean, measured conc.)
1000 mg/l (loading rate); 1.52 mg/l (arithmetic mean, measure conc.)
Details on test conditions:
Test vessels:
Test vessels were 200 ml conical glass flasks fitted with glass stoppers to provide an airtight seal. The vessels and stoppers were cleaned and sterilised prior to use (autoclaving). Two sterile glass marbles were placed in each flask to aid mixing during incubation of the test cultures.
Prior to the test, the vessels were rinsed with the appropriate WAF solution in order to saturate the surface.

Acute toxicity test:
The algae were exposed for 72 h to graded concentrations of the test substance (WAF) and a control under static conditions. The starting cell concentration was 1000 cells/ml and the test vessels were completely full and sealed. The purpose of the decreased cell number (OECD 201: 10000 cells/ml) was to prevent the development of a large biomass by the end of the study. This was necessary in the sealed systems because of the risk of depleting the C02 source and reducing the carbonate concentration to overwhelm the buffering capacity of the medium, and allowing the pH to shift to alkaline levels. This effect would have artificially limited the growth rate.
The cell concentrations were determined at the start of the test in the application algae­ suspension and after 24, 48 and 72 h. The volume of test solution removed for evaluation and that displaced by the stopper were replaced using spare WAF which had been stored in a sealed vessel. A separate spare vessel was prepared for each replacement interval (1 vessel per test concentration). The reason for replacement of removed test solution with fresh WAF was to prevent re-equilibration of the volatile test substance from the WAF to the head-space above the WAF, ensuring constant concentration of the WAF.
The culture vessels were incubated at 22 ± 1 °C with a light intensity adjusted to 8000 Lux (= 120 uE/m2s) ± 20%. prior to the test and during the test. The cultures were resuspended continuously by shaking on a laboratory shaker. The cell density was measured daily using an electronic particle counter.
In the controls and WAFs, pH values were measured directly before adding the algae and at the end of the test. This parameter was not measured in the test vessels due to possible absorption of the surface film on the electrodes. For measuring the pH values and assessing the stability of the substance, samples were taken from the WAF parallel to the filling of the test vessels at test start and from the test vessels at test termination.

The following loading rates were tested:
control (3 replicates)
100 mg/l (3 replicates)
1000 mg/l (5 replicates)

The performance of the enhanced limit test was chosen (according to CONCAWE 1992), because a pre-test finding indicated that a WAF prepared with a loading of 1000 mg/L was not toxic.

Statistics
NOEC and LOEC values were determined using one-way ANOVA followed by Williams' Multiple Sequential t-Test using programme ToxRat V 1.06.


Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 1.52 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.52 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate

Any other information on results incl. tables

Stability of WAFs - Results of Chemical Analysis

The results of chemical analysis are given in Table 1 (below)

Table 1 Concentration of Zonyl® in selected samples.

Sampling time

Loading mg/l

Sample identification

Concentration of PFBE mg/l

mean

1

2

3

4

Day 0

control

a

<0.05

<0.05

 

 

<0.05

23/01/2001

100

b

1.01

1.02

0.78

0.86

0.92

 

1000

c

1.50

-

1.46

1.61

1.52

Day 3

control

1

<0.05

<0.05

 

 

<0.05

26/01/2001

100

4

<0.05

<0.05

<0.05

 

<0.05

 

100

5

<0.05

<0.05

<0.05

 

<0.05

 

100

6

<0.05

<0.05

<0.05

 

<0.05

 

1000

7

0.11

0.10

0.11

 

0.11

 

1000

8

<0.05

<0.05

<0.05

 

<0.05

 

1000

9

<0.05

<0.05

<0.05

 

<0.05

 

1000

10

<0.05

<0.05

<0.05

 

<0.05

 

1000

11

<0.05

<0.05

<0.05

 

<0.05

At the start of the test, PFBE concentrations were analysed to be 0.92 mg/L in the WAF loaded with 100 mg/L and 1.52 mg/L in the WAF with the loading rate of 1000 mg/L.. At the end of the test, PFBE concentrations decreased below the limit of detection except for 1 vessel (no 7). Because the test vessels had to be opened daily for taking samples, the decline of the PFBE concentrations can be attributed to the evaporation of the high volatile substance during the sampling procedure. The concentrations at the start of the test were used for correlating the growth inhibition effects to analytical concentrations,

Algal growth inhibition test

The development of the cell number over time is summarised in Table 2. Computed biomass integral values are listed in Table 3. Growth rates are summarised in Table 4.

The loading rates of 100 mg/L and 1 000 mg/L or measured concentrations of 0.92 mg/L and 1.52 mg/L, respectively, had no significant inhibitory effect on biomass development and growth rate after 72 h compared to the controls. After 72 h, a promotion of growth was observed at the highest loading rate of 1 000 mg/L.

Because no significant inhibition of growth (B) and growth rate (r) was observed compared to the controls after 72 h, effect values are as follows:

NOEC(B, r) >/= 1000 mg/l (loading rate)

LOEC(B, r) > 1000 mg/l (loading rate)

EC50(B, r) >1000 mg/l (loading rate)

NOEC(B, r) >/= 1.5 mg/l (measured conc)

LOEC(B, r) > 1.5 mg/l (measured conc)

EC50(B, r) >1.5 mg/l (measured conc)

Table 2. Cell Number of the algae cultures as dependent on loading rate of the test substance and time. Mean = arithmetic mean; Std. Dev = standard deviation; n=number of replicates.

Loading mg/l

Measured conc mg/l

 

0 hr

24 hr

48 hr

72 hr

Control

0

mean

1000

2426

10933.3

44626

 

 

Std. Dev

0.00

402

1339

3762

 

 

n

3

3

3

3

100

0.92

mean

1000

2033

14216.7

48276

 

 

Std. Dev

0.00

350

4102

3170

 

 

n

3

3

3

3

1000

1.52

mean

1000

1702

12028.0

68080*

 

 

Std. Dev

0.00

151

1875

8261

 

 

n

5

5

5

5

*significant increase compared to control p<0.05

Table 4. Inhibition of biomass integral: values of the response variable and % inhibition as computed from the raw data for various experimental test intervals inspected. (B = Computed value of the biomass integral, -%inhibition = increase in growth relative to that of control.

Loading mg/l

Measured mg/l

0-24 hr

0-48 hr

0-72 hr

 

 

B

%

B

%

B

%

Control

0

17120

0.0

153440

0.0

796160

0.0

100

0.92

12400

27.6

183400

-19.5

909320

-14.2

1000

1.52

8424

50.8

149184

2.8

1086480

-36.5*

* significant increase compared to control p<0.05

Table 5: Inhibition of growth rate: values of the response variable and % inhibition as computed from the raw data for various experimental test intervals inspected. (B = computed value of the Biomass Integral; -% inhibition = increase in growth relative to that of control.

Treatment mg/l

Measured mg/l

0-24 hr

0-48 hr

0-72 hr

 

 

G

%I

G

%I

G

%I

Control

0

0.88

0.0

1.19

0.0

1.27

0.0

100

0.92

0.70

20.3

1.31

-9.9

1.29

-2.1

1000

1.52

0.53

39.7

1.24

-3.8

1.4

-11.0

Validity of the test

The conditions for the validity of the test are complied according to OECD Test Guideline 201:

The cell concentration in the control cultures did increase by a factor of >16 within three days.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
any other information on results incl. tables
Conclusions:
Summary of the test results

Inhibition of growth

Up to the highest nominal loading rate of 1000 mg/L PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene), no significant inhibition of growth (biomass) and growth rate was observed after 72 h compared to controls.


NOEL >/= 1000 mg/L (loading rate)
LOEL > 1000 mg/L (loading rate )
EL50 > 1000 mg/L (loading rate)

NOEL >/= 1.52 mg/L (lmeasured conc)
LOEL > 1.52 mg/L (measured conc )
EC50 > 1.52 mg/L (measured conc)


Promotion of growth
A promotion of growth (biomass) of 36.5 % could be observed at the highest loading rate of 1000 mg/L after 72 h.


Executive summary:

The objective of the study was the assessment of the acute toxicity of PFBE on the green alga Selenastrum capricornutum. Due to the low water solubility and high volatility of the test substance, the study was performed using water accommodated fractions (WAF) of PFBE as the test medium, as recommended for ecotoxicological testing of petroleum products by CONCAWE 1992 and for aquatic toxicity testing of lubricants by ASTM (standard protocol 06081-97). These protocols are designed to comply with the general principles outlined in the respective OECD guidelines.

The growth of the algae under static conditions was assessed after 24, 48, and 72 hours. The validity criteria of the OECD Test Guideline 201 are fulfilled under the conditions of the modified procedures.

Summary of the test results

Inhibition of growth

Up to the highest nominal loading rate of 1000 mg/L PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene), no significant inhibition of growth (biomass) and growth rate was observed after 72 h compared to controls.

NOEC >/= 1000 mg/L (loading rate)

LOEC > 1000 mg/L (loading rate )

EC50 > 1000 mg/L (loading rate)

NOEC >/= 1.52 mg/L (measured conc)

LOEC > 1.52 mg/L (measured conc )

EC50 > 1.52 mg/L (measured conc)

Promotion of growth

A promotion of growth (biomass) of 36.5 % could be observed at the highest loading rate of 1000 mg/L after 72 h.