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Long-term toxicity to fish

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Reference
Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-07-27 to 2010-08-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The fractions C20 and C22 of C20/22 ATQ trocken and the control were analytically verified from alternating test replicates in 3 sampling intervals from freshly prepared and 48 h old test solutions (last interval 24 h). Stock solutions were additionally analysed.
- Sampling method: The sorption of the test item on glass was quantified. Separate test vessels were set up and used for the different sampling dates and concentrations.
The stock solution vessel and the test replicates were rinsed (stock solution vessel twice) with appropriate volumes of acetonitrile. The concentration of the test item in these solutions was measured and the adsorbed test item amount will calculated from this concentration.
Before this procedure the vessels were carefully rinsed with dechlorinated water to remove all test item dissolved in remaining test media.

- Sample storage conditions before analysis:All samples were stored at room temperature before preparation and before analysis.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 5.00 mg/L was prepared.
- Eluate: Natural river water
- Differential loading: 3.20 – 1.00 – 0.32 – 0.10 – 0.032 mg/L.
- Controls: River water as dilution water (without test item)
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Gnathostoma, Pisces, Osteichthyes, Teleostei, Clupeiformes, Cyprinidae
- Source: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, D-31135 Hildesheim, An der Scharlake 39, Germany. All fish eggs used in the test were gained at DR.U.NOACK-LABORATORIEN from a single brood stock.


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Adult zebrafish were kept in separate aquaria. About 15 minutes before start of artificial dawning (1 h) rectangular dishes (26 cm x 14 cm x 6 cm), covered with a stainless steel mesh and provided with artificial plants, were introduced into the aquaria. At the end of dawning the glass dishes were gently removed. At least 300 eggs were taken and immediately distributed to the test solutions and dilution water (for control).
- Subsequent handling of eggs: After approximately 2 h eggs were checked for fertilization. Under a stereo microscope every embryo was checked for its blastomer phase. Eggs with only a 2 cell blastomer were regarded not to be fertilised. These eggs as well as coagulated eggs were discarded. Only fertilised eggs with more than 2 cells were introduced in the test vessels. 10 eggs were introduced per replicate.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
9 d
Hardness:
Total hardness 102 mg CO3/L (beginning of the test)
Test temperature:
Water Temperature in the Test Media

Temperature [°C]
Geometric mean measured test concentrations [mg/L]
Study day Control 0.0246 0.0753 0.243 0.762 1.62
0 24.0 24.2 24.3 24.3 24.3 24.4
1 24.0 24.1 24.0 24.1 24.1 24.1
2 old 24.1 24.2 24.3 24.4 24.4 24.5
new 24.4 24.4 24.4 24.4 24.6 25.1
3 24.8 24.9 24.9 24.8 24.9 24.9
4 old 24.1 24.0 24.1 24.1 24.1 24.2
new 24.2 24.1 24.0 24.1 24.2 -
5 24.1 24.0 24.0 24.0 24.0 -
6 old 24.4 24.3 24.2 24.2 24.1 -
new 24.5 24.2 24.1 24.3 24.3 -
7 24.6 24.4 24.4 24.5 24.5 -
8 old 24.5 24.5 24.7 24.6 24.8 -
new 24.6 24.7 24.6 24.7 24.7 -
9 24.5 24.4 24.2 24.4 24.5 -
Mean 24.3 24.3 24.3 24.4 24.4 24.5
SD ± 0.256 0.260 0.277 0.241 0.284 0.393
Min. 24.0 24.0 24.0 24.0 24.0 24.1
Max. 24.8 24.9 24.9 24.8 24.9 25.1
pH:
pH-value
Geometric mean measured test concentrations [mg/L]
Study day Control 0.0246 0.0753 0.243 0.762 1.62
0 7.90 7.84 7.83 7.81 7.82 7.83
1 7.83 7.96 7.97 7.97 7.91 7.91
2 old 7.93 7.97 7.97 7.95 7.93 7.92
new 7.88 7.84 7.84 7.86 7.88 7.90
3 7.93 8.02 8.02 8.02 8.02 8.03
4 old 7.64 7.73 7.70 7.69 7.65 7.71
new 7.51 7.71 7.66 7.69 7.69 -
5 8.03 8.13 8.13 8.09 8.07 -
6 old 8.04 8.09 8.10 8.05 8.04 -
new 8.03 8.04 8.06 8.04 8.03 -
7 8.10 8.11 8.10 8.02 8.00 -
8 old 8.09 8.09 8.07 8.03 8.04 -
new 8.02 8.06 8.06 8.05 7.98 -
9 8.04 8.08 8.10 8.03 7.97 -
Mean 7.93 7.98 7.97 7.95 7.93 7.88
SD ± 0.171 0.141 0.155 0.134 0.131 0.107
Min. 7.51 7.71 7.66 7.69 7.65 7.71
Max. 8.10 8.13 8.13 8.09 8.07 8.03
Dissolved oxygen:
Dissolved Oxygen in Percent Air Saturation Value

Dissolved Oxygen [%]
Geometric mean measured test concentrations [mg/L]
Study day Control 0.0246 0.0753 0.243 0.762 1.62
0 100 100 100 100 100 100
1 100 100 100 100 100 100
2 old 100 100 100 100 100 100
new 100 100 100 100 100 100
3 100 100 100 100 100 100
4 old 100 100 100 100 100 100
new 100 100 100 100 100 -
5 100 100 100 100 100 -
6 old 100 100 100 100 100 -
new 100 100 100 100 100 -
7 100 100 100 100 100 -
8 old 100 100 100 100 100 -
new 100 100 100 100 100 -
9 100 100 100 100 100 -
Mean 100 100 100 100 100 100
SD ± 100 100 100 100 100 100
Min. 100 100 100 100 100 100
Max. 100 100 100 100 100 100
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Crystallisation dishes (inner diameter 13.5 cm, water height about
5 cm) were used. The volume of the test media in the dishes was about 500 mL.
- Aeration: No aeration was provided. Semi-static conditions with renewal of the test media in 48 h intervals was performed.
- No. of fertilized eggs/embryos per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Biomass loading rate: Mean dry weight of control fish 0.477 mg in 0.5 L test medium


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: C20/22 ATQ is a quaternary ammonium salt and belongs to the group of cationic surfactants. Cationic surfactants often settle on glass surfaces when tested in synthetic water. Using river water with dissolved organic carbon and suspended matter often overcomes the testing issues described before. For cationic surfactants like long-chain primary alkyl amines strong sorption to DOC and suspended matter of the river water occurs but sorption to the glass surface can be neglected. That means the test substance is available in the test system for exposure of the test organism either in dissolved, sorbed to DOC and sorbed to suspended matter.
Water of the river Böhme was used. The sampling site is located in Bad Fallingbostel.
River Böhme
Location Fischendorfer Straße,
29683 Bad Fallingbostel, Germany,
52°53`51.81 N / 9°45`41.44 E
Sampling Date 2010-06-02
Weather on Day of Sampling sunny, ca. 22 °C
Colour brownish, clear
pH 7.79
Conductivity [µS/cm] 452
Dissolved Oxygen [mg O2/L] 8.93
DOC [mg C/L] 6.96
TOC [mg C/L] 6.61
Ammonium-N [mg N/L] 0.065
Nitrate-N [mg N/L] 1.35
Total Nitrogen [mg N/L] 2.64
o-Phosphate-p [mg P/L] 0.063
Total Phosphate [mg P/L] 0.082
Suspended Matter [mg/L] 9.5
Total Hardness [mg CO3/L] 116
Storage conditions 6 ± 2 °C




OTHER TEST CONDITIONS
- pH: 6 – 8
- Photoperiod: 16 h photoperiod daily
- Light intensity: 0.1 - 10 µmol photons • m-2 • s-1on water surface (diffuse light)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Hatched eggs The number of hatched eggs were determined daily until 5 days after post-hatch.

Post hatch period Per definition the post hatch period begun when at least 80 % of all fertilized and living embryos in the co ntrol group have hatched (day 4 of the study).

Mortality Criteria for mortality vary according to life stage:

For eggs: Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, were checked daily. Dead eggs were discarded.

For embryos: Absence of body movement and/or heart-beat, change in coloration. Dead embryos were discarded.

For larvae: Immobility and/or absence of respiratory movement and/or absence of heart-beat (as far as visible) and/o r lack of reaction to mechanical stimulus.
Abnormal appearance and behaviour were also be recorded.
The number of larvae or fish showing abnormality of body form and/or pigmentation and the stage of yolk-sac absorption were recorded. Abnormal animals were only removed from the test vessels on death. Abnormalities, e.g. hyperventilation, uncoordinated swimming, swim-up behaviour, atypical quiescence, and atypical feeding behaviour were also be recorded by visually inspecting each replicate.
At the end of exposure (post-hatch day 5) the fish were euthanized in a Benzocaine solution and the total length of all survivors were measured to the nearest 0.001 mm.
A pooled fish dry weight of each test replicate was determined from all survivors at the end of exposure (post-hatch day 5).
The fish were dried for 24 h at 60 °C. Dry biomass weight was measured to the nearest 0.001 mg.


VEHICLE CONTROL PERFORMED: no


Key result
Duration:
9 d
Dose descriptor:
NOEC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: OVERALL: Hatch, Growth & Fry survival
Duration:
9 d
Dose descriptor:
LOEC
Effect conc.:
0.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: OVERALL: Hatch, Growth & Fry survival
Duration:
9 d
Dose descriptor:
LC50
Effect conc.:
1.03 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (0.776 – 1.40)
Details on results:
- Mortality/survival at embryo and larval stages: The post hatch success in all control replicates met the guideline criteria. The fry survival (post hatch success) at the end of the study was 100 % in the control group and in the test concentrations of 0.0246 – 0.243 mg/L, 96 % in the test concentration of 0.614 mg/l and 0 % in the highest test item concentration of 1.62 mg/L.
One way analysis of variance and Dunnett’s test were carried out for the results of post hatch day 5 (end of the study). A statistically significant difference was found for the highest concentration (1.62 mg/L).

- Overall mortality/survival: Overall survival at the end of the study was 100 % in the control group and in the test concentrations of 0.0246 – 0.243 mg/L, 87 % in the test concentration of 0.762 mg/l and 0 % in the highest test item concentration of 1.62 mg/L.
One way analysis of variance and Dunnett’s test were carried out for the results of overall survival on day 5 (end of the study). Statistically significant differences were found for the concentrations 0.762 and 1.62 mg/L.

- Days to hatch and numbers hatched: Egg hatch began on study day 3 in the control and the test item concentrations 0.0246, 0.753 and 0.762 mg/L and continued until study day 5. Study day 4 was determined to be post hatch day 0 (PHD 0) with a control hatching rate of 90 %.
Statistical procedures (one way analysis of variance) were applied for study days 4 and 5 (post hatch days 0 and 1). Statistically significant differences were found on post hatch days 0 and 1 for the highest test concentration (1.62 mg/L). On post hatch day 0 significant differences were also found for the concentrations 0.0246 and 0.062 mg/L. However, these effects were transient and considered not biologically significant.

- Data for length and weight of surviving fish: The fry growth, expressed as length and dry weight, was measured on study day 9 (PHD 5).
One way analysis of variance and Dunnett’s test were carried out for the results of PHD 5 (end of the study). For the length data the detected statistical differences were considered biologically not being significant. A statistically significant difference of the weight was found for the concentrations 0.762 mg/L

- Type of and number with morphological abnormalities: No biologically significant morphological and behavioural effects were observed in any tested replicate (except the highest tested concentration with 100 % mortality, no further data presented).

Reported statistics and error estimates:
One way analysis of variance (ANOVA) were used for NOEC/LOEC calculations. When running a one way analysis of variance a normality test and an equal variance test were done first. For the parameters hatch, growth (length and dry weight), post hatch survival and overall fry survival (mortality), the following statistical tests were conducted:
Hatching data of study day 4 (post hatch day 0) and study day 5 (post hatch day 1) were analysed with ANOVA.
Dry weight data, length data, post hatch survival and overall survival (mortality) data of study day 9 (post hatch day 5) were analysed with ANOVA. Normality tests for mortality and post hatch success failed. No transformations were carried out with these data because the data sets were not estimated to follow a normal distribution.
These statistical analyses were conducted with conclusions of statistical significance based on a 95 % confidence level. The a-value (acceptable probability of incorrectly concluding that there is a difference) was 0.05.

Overall Survival and Mortality on Study Day 9 (Post Hatch Day 5)

Geometric mean measured test item conc.

[mg/L]

Replicate

Live fry on post hatch
day 5 (Study day 9)

n/10

Overall survival

 [%]

Mortality

 [%]

Control

1

10

100

0

2

10

100

0

3

10

100

0

Mean

10

100

0

0.0246

1

10

100

0

2

10

100

0

3

10

100

0

Mean

10

100

0

0.0753

1

10

100

0

2

10

100

0

3

10

100

0

Mean

10

100

0

0.243

1

10

100

0

2

10

100

0

3

10

100

0

Mean

10

100

0

0.762

1

8

80

20

2

9

90

10

3

9

90

10

Mean

9

87

     13 (+)

1.62

1

0

0

100

2

0

0

100

3

0

0

100

Mean

0

0

     100 (+)

Post Hatch Survival on Study Day 9 (Post Hatch Day 5)

                      Live fry on study day 5 were set to 100 % for the calculation of the post hatch survival

Geometric mean measured test item conc.

[mg/L]

Replicate

Live fry on study day                                     n/10

Post Hatch survival

[%]

Post hatch
day 0
(Study day 4)

Post hatch
day 1
(Study day 5)

Post hatch
day 5
(Study day 9)

Control

1

10

10

10

100

2

9

10

10

100

3

8

10

10

100

Mean

9.0

10.0

10.0

100

0.0246

1

4

10

10

100

2

3

10

10

100

3

4

10

10

100

Mean

3.7

10.0

10.0

100

0.0753

1

6

10

10

100

2

5

10

10

100

3

7

10

10

100

Mean

6.0

10.0

10.0

100

0.243

1

8

10

10

100

2

8

10

10

100

3

8

10

10

100

Mean

8.0

10.0

10.0

100

0.762

1

9

9

8

89

2

9

9

9

100

3

7

9

9

100

Mean

8.3

9.0

8.7

 96

1.62

1

0

0

0

0

2

0

0

0

0

3

0

0

0

0

Mean

0.0

0.0

0.0

     0 (+)

Egg Hatch / Hatching Time

Geometric mean measured test item conc.

[mg/L]

Replicate

Egg hatch [%]

Post hatch
day -2
(Study day 2)

Post hatch
day -1
(Study day 3)

Post hatch
day 0
(Study day 4)

Post hatch
day 1
 
(Study day 5)

Post hatch
day 2
  
(Study day 6)

Control

1

0

70

100

100

100

2

0

30

90

90

100

3

0

0

80

100

100

Mean

0

33

90

97

100

0.0246

1

0

10

40

100

100

2

0

10

30

100

100

3

0

0

40

90

100

Mean

0

7

    37 (+)

97

100

0.0753

1

0

0

60

100

100

2

0

10

50

100

100

3

0

0

70

100

100

Mean

0

3

     60 (+)

100

100

0.243

1

0

0

80

100

100

2

0

0

80

100

100

3

0

0

80

100

100

Mean

0

0

80

100

100

0.762

1

0

50

90

90

90

2

0

50

90

90

90

3

0

30

70

90

90

Mean

0

43

83

90

90

1.62

1

0

0

0

0

0

2

0

0

0

0

0

3

0

0

0

0

0

Mean

0

0

     0 (+)

     0 (+)

0

Validity criteria fulfilled:
yes
Conclusions:
C20/22 ATQ trocken caused significant effects on short-term toxicity test with embryo and sac-fry stages of zebrafish with natural river Water up to the dosage level of 0.762 mg/L (geometric mean measured test concentration). Based on the parameters hatch, growth (expressed as length and dry weight), post hatch and overall survival (mortality) the overall NOEC (post hatch day 0 – 5) was 0.243 mg/L (geometric mean measured test concentration). The overall LOEC was 0.762 mg/L (geometric mean measured test concentration).
Executive summary:

The effects of the test item C20/22 ATQ trocken to the embryo and sac-fry stages of fish (Zebrafish / Danio rerio) were determined according to OECD Guideline 212 from 2010-07-27 to  2010-08-20 with an definitive exposure phase from 2010-07-27 to 2010-08-05 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A semi-static test procedure with renewal of the test media in 48 h intervals was performed with the nominal concentrations of 0.032 - 0.100 - 0.320 - 1.00 - 3.20 mg/L, corresponding to geometric mean measured concentrations of 0.0246 – 0.0753 – 0.243 – 0.762 – 1.62 mg/L.

The test was started by placing fertilized eggs in the test vessels and lasted 9 days (5 days post-hatch). 30 eggs of Danio rerio were exposed to the test concentrations and the control (3 replicates with 10 eggs each).

On day four 90 % of the control larvae had hatched. Therefore, study day 4 had been defined as post hatch day 0 (= PHD 0).

Different toxic endpoints have been determined: egg hatch, time to hatch, fry growth (expressed as length and weight), morphological and behavioural effects, post hatch survival, overall fry survival and mortality, respectively. The results of the named parameters were checked for statistically significant differences. The NOEC, LOEC and LC-values were determined based on the statistical results. The revealing results are summarized in Table 1 and Table 2.

The concentration of the test item and control were analytically verified via LC-MS/MS from freshly prepared media on days 0, 4 and 8 and from 48 hours old media on days 2, 6 and 9.

The measured concentrations of C20/22 ATQ trocken (C20 and C22 fraction) from the fresh test media were in the range of 91 – 129 %. The measured aqueous concentrations after the first media interval (after 48 hours) showed a decrease of the measured concentrations (24 – 47 % of the nominal values). However, the measured aqueous concentrations from the following renewal intervals (48 hours and 24 hours old solutions, respectively of day 6 and 9) were higher. Together with the results of the measured amounts from glass surfaces these data show that the adsorption effects in the first quarter of the study were clearly higher than in the remaining part of the study. It is supposed that adsorption effects took place very quickly whereas in the remaining part of the study the glass surfaces were saturated at a higher grade and therefore adsorbed less C20/22 ATQ trocken. When basing all recovery calculations on the actual amounts applied (considering 5 renewals leading to 5 times of a simple application amount) the amount of adsorbed C20/22 ATQ trocken is less than 25 % of the total applied C20/22 ATQ trocken. Finally it can be assumed that the bioavailable concentrations from day 2 on (after the first renewal) were higher than during the first 48 hours of exposure.

The test item has a low water solubility and sorbs to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item is difficult to test in synthetic water (e.g. sorption to the test organism and walls of the test vessel) and results from such tests depend from the test settings applied. Using natural river water which contains particulate as well as dissolved organic carbon

to which the test item can sorb partially reduces the difficulties encountered in tests with synthetic water e.g. preventing that the test item settles onto surfaces. The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries. Due to the short exposure period these low recoveries cannot be associated to biodegradation. This means the test substance is present in the test system and therefore available for exposure (dissolved in water and sorbed also called bulk). This so called Bulk Approach is described by ECETOC (2003). Due to the properties of the test item geometric mean measured concentrations have to be used (see Table 1).

Table 1:       Hatch, Growth, Fry survival: NOEC, LOEC

                      based on geometric mean measured test item concentrations of C20/22 ATQ trocken [mg/L]

Parameter

NOEC

LOEC

Hatch

0.762

   1.62

Length

0.762

   1.62

Weight

0.243

   0.762

Post hatch survival

0.762

   1.62

Overall survival

0.243

   0.762

Overall NOEC and LOEC

0.243

   0.762

Table 2 :      NOEC and LC-Values with 95 % Confidence Interval on Study Day 9 (Post Hatch Day 5)

                      based on geometric mean measured test item concentrations of C20/22 ATQ trocken [mg/L]

LC50

1.03

(95 % Confidence Interval)

(0.776 – 1.40)

  LC100

1.62

Lowest test item concentration

with 100 % mortality on Study day 9

LC0

0.243

Highest test item concentration

with 0 % mortality on Study day 9

Description of key information

C20/22 ATQ trocken caused significant effects on short-term toxicity test with embryo and sac-fry stages of zebrafish with natural river Water up to the dosage level of 0.762 mg/L (geometric mean measured test concentration). Based on the parameters hatch, growth (expressed as length and dry weight), post hatch and overall survival (mortality) the overall NOEC (post hatch day 0 – 5) was 0.243 mg/L (geometric mean measured test concentration). The overall LOEC was 0.762 mg/L (geometric mean measured test concentration). 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.24 mg/L

Additional information

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